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1.
Anal Chim Acta ; 1154: 338310, 2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33736798

RESUMO

Novel coronavirus disease (COVID-19) caused by SARS-CoV-2 is an ongoing global pandemic associated with high rates of morbidity and mortality. RT-qPCR has become the diagnostic standard for the testing of SARS-CoV-2 in most countries. COVID-19 diagnosis generally relies upon RT-qPCR-mediated identification of SARS-CoV-2 viral RNA, which is costly, labor-extensive, and requires specialized training and equipment. Herein, we established a novel one-tube rapid diagnostic approach based upon formamide and colorimetric RT-LAMP (One-Pot RT-LAMP) that can be used to diagnose COVID-19 without the extraction of specific viral RNA. The technique could visually detect SARS-CoV-2 within 45 min with a limit of detection of 5 copies per reaction in extracted RNA, and about 7.66 virus copies per µL in viral transport medium. The One-Pot RT-LAMP test showed a high specificity without cross-reactivity with 12 viruses including SARS-CoV, MERS-CoV, and human infectious influenza virus (H1N1/H3N2 of influenza A and B virus, ect. We validated this One-Pot RT-LAMP approach by its successful use for the analysis of 45 clinical nasopharyngeal swab samples, yielding results identical to those of traditional RT-qPCR analyses, while achieving good selectivity and sensitivity relative to a commercial RT-qPCR approach. As such, this One-Pot RT-LAMP technology may be a valid means of conducting high-sensitivity, low-cost and rapid SARS-CoV-2 identification without the extraction of viral RNA.


Assuntos
/métodos , /diagnóstico , /virologia , Primers do DNA/química , Primers do DNA/metabolismo , Humanos , Limite de Detecção , Técnicas de Diagnóstico Molecular , Nasofaringe/virologia , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/análise , RNA Viral/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , /isolamento & purificação , Fatores de Tempo
2.
Nat Commun ; 12(1): 804, 2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33547322

RESUMO

Evolution of xeno nucleic acid (XNA) world essentially requires template-directed synthesis of XNA polymers. In this study, we demonstrate template-directed synthesis of an acyclic XNA, acyclic L-threoninol nucleic acid (L-aTNA), via chemical ligation mediated by N-cyanoimidazole. The ligation of an L-aTNA fragment on an L-aTNA template is significantly faster and occurs in considerably higher yield than DNA ligation. Both L-aTNA ligation on a DNA template and DNA ligation on an L-aTNA template are also observed. High efficiency ligation of trimer L-aTNA fragments to a template-bound primer is achieved. Furthermore, a pseudo primer extension reaction is demonstrated using a pool of random L-aTNA trimers as substrates. To the best of our knowledge, this is the first example of polymerase-like primer extension of XNA with all four nucleobases, generating phosphodiester bonding without any special modification. This technique paves the way for a genetic system of the L-aTNA world.


Assuntos
Amino Álcoois/metabolismo , Butileno Glicóis/metabolismo , DNA/genética , Imidazóis/química , Ácidos Nucleicos/síntese química , RNA/genética , Amino Álcoois/química , Pareamento de Bases , Biocatálise , Butileno Glicóis/química , Cátions Bivalentes , DNA/química , DNA/metabolismo , Primers do DNA/química , Primers do DNA/metabolismo , Manganês/química , Manganês/metabolismo , Conformação de Ácido Nucleico , RNA/química , RNA/metabolismo , Soluções
3.
Methods Mol Biol ; 2225: 77-92, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33108658

RESUMO

Adeno-associated virus (AAV) is a helper-dependent single-stranded DNA parvovirus. Over the years, AAV has become the vector of choice in the gene therapy field due to its safety profile and low immunogenicity. With a carrying capacity of 4.2 kbp, these vectors have demonstrated their clinical value, especially in the field of ophthalmology. Herein we describe methods for the molecular design and packaging of AAV viral vectors. These methods apply to the design of single-stranded or self-complementary AAV vectors.


Assuntos
Clonagem Molecular/métodos , Dependovirus/genética , Engenharia Genética/métodos , Transgenes , /genética , Primers do DNA/química , Primers do DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Dependovirus/metabolismo , Escherichia coli/virologia , Terapia Genética/métodos , Humanos , Plasmídeos/química , Plasmídeos/metabolismo , Transdução Genética
4.
PLoS One ; 15(12): e0244882, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33382861

RESUMO

SARS-CoV-2 testing is crucial to controlling the spread of this virus, yet shortages of nucleic acid extraction supplies and other key reagents have hindered the response to COVID-19 in the US. Several groups have described loop-mediated isothermal amplification (LAMP) assays for SARS-CoV-2, including testing directly from nasopharyngeal swabs and eliminating the need for reagents in short supply. Frequent surveillance of individuals attending work or school is currently unavailable to most people but will likely be necessary to reduce the ~50% of transmission that occurs when individuals are nonsymptomatic. Here we describe a fluorescence-based RT-LAMP test using direct nasopharyngeal swab samples and show consistent detection in clinically confirmed primary samples with a limit of detection (LOD) of ~625 copies/µl, approximately 100-fold lower sensitivity than qRT-PCR. While less sensitive than extraction-based molecular methods, RT-LAMP without RNA extraction is fast and inexpensive. Here we also demonstrate that adding a lysis buffer directly into the RT-LAMP reaction improves the sensitivity of some samples by approximately 10-fold. Furthermore, purified RNA in this assay achieves a similar LOD to qRT-PCR. These results indicate that high-throughput RT-LAMP testing could augment qRT-PCR in SARS-CoV-2 surveillance programs, especially while the availability of qRT-PCR testing and RNA extraction reagents is constrained.


Assuntos
Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , RNA Viral/genética , /genética , /diagnóstico , Primers do DNA/química , Primers do DNA/genética , Humanos , Limite de Detecção , Nasofaringe/virologia
5.
Anal Chem ; 92(20): 14139-14144, 2020 10 20.
Artigo em Inglês | MEDLINE | ID: mdl-32967427

RESUMO

The infection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that causes the coronavirus disease 2019 (COVID-19) has threatened public health worldwide. The easy human-to-human transmission of this virus has rapidly evolved into a global pandemic. Therefore, to control the community spread of the virus, it is crucial to identify the infected individuals, including asymptomatic people. Hence, a specific and rapid assay is crucial for the early diagnosis and active monitoring of individuals potentially exposed to SARS-CoV-2 for controlling the COVID-19 outbreak. In this study, we have developed the novel lateral flow strip membrane (LFSM) assay that allows the simultaneous detection of RdRp, ORF3a, and N genes using the PCR product obtained by using the single-tube reverse transcription polymerase chain reaction (RT-PCR). The LFSM assay allows detection of SARS-CoV-2 in 30 min at 25 °C after the RT-PCR with the detection limit of 10 copies/test for each gene. The clinical performance of the LFSM assay for the detection of SARS-Cov-2 was evaluated using 162 clinical samples previously detected by using the commercial assay. The percent positive agreement, percent negative agreement, and overall percent agreement of the LFSM assay with the commercial assay were 100% (94.2-100%), 99.0% (94.6-100%), and 99.4% (96.6-100%), respectively. Therefore, the results of the LFSM assay showed significantly high concordance with the commercial assay for the detection of SARS-CoV-2 in clinical specimens. Therefore, we conclude that the developed LFSM assay can be used alone or complementary to the RT-PCR or other methods for the diagnosis and monitoring of the patients to curb community transmission and the pandemic.


Assuntos
Betacoronavirus/genética , Fluorometria/métodos , Proteínas do Nucleocapsídeo/análise , Proteínas Virais Reguladoras e Acessórias/análise , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/virologia , Primers do DNA/química , Primers do DNA/metabolismo , Corantes Fluorescentes/química , Fluorometria/instrumentação , Humanos , Limite de Detecção , Técnicas de Amplificação de Ácido Nucleico , Proteínas do Nucleocapsídeo/genética , Proteínas do Nucleocapsídeo/metabolismo , Pandemias , Fosfoproteínas , Pneumonia Viral/diagnóstico , Pneumonia Viral/virologia , /metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Virais Reguladoras e Acessórias/genética , Proteínas Virais Reguladoras e Acessórias/metabolismo
6.
Exp Parasitol ; 217: 107960, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32755552

RESUMO

Guinea worm Dracunculus medinensis causes debilitating disease in people and is subject to an ongoing global eradication programme. Research and controls are constrained by a lack of diagnostic tools. We developed a specific and sensitive LAMP method for detecting D. medinensis larval DNA in copepod vectors. We were able to detect a single larva in a background of field-collected copepods. This method could form the basis of a "pond-side test" for detecting potential sources of Guinea worm infection in the environment, in copepods, including in the guts of fish as potential transport hosts, enabling research, surveillance and targeting of control measures. The key constraint on the utility of this assay as a field diagnostic, is a lack of knowledge of variation in the temporal and spatial distribution of D. medinensis larvae in copepods in water bodies in the affected areas and how best to sample copepods to obtain a reliable diagnostic sample. These fundamental knowledge gaps could readily be addressed with field collections of samples across areas experiencing a range of worm infection frequencies, coupled with field and laboratory analyses using LAMP and PCR.


Assuntos
Copépodes/parasitologia , Dracunculus/isolamento & purificação , Técnicas de Diagnóstico Molecular/normas , Técnicas de Amplificação de Ácido Nucleico/normas , Tanques/parasitologia , África , Animais , Sequência de Bases , Gatos , Copépodes/genética , Primers do DNA/química , DNA de Helmintos/isolamento & purificação , Vetores de Doenças , Cães , Dracunculus/genética , Humanos , Papio , Sensibilidade e Especificidade , Fatores de Tempo
7.
Arch Virol ; 165(10): 2335-2340, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32719956

RESUMO

Sapoviruses are increasingly being recognized as pathogens associated with gastroenteritis in humans. Human sapoviruses are currently assigned to 18 genotypes (GI.1-7, GII.1-8, GIV.1, and GV.1-2) based on the sequence of the region encoding the major structural protein. In this study, we evaluated 11 polymerase chain reaction (PCR) assays using published and newly designed/modified primers and showed that four PCR assays with different primer combinations amplified all of the tested human sapovirus genotypes using either synthetic DNA or cDNA prepared from human sapovirus-positive fecal specimens. These assays can be used as improved broadly reactive screening tests or as tools for molecular characterization of human sapoviruses.


Assuntos
Infecções por Caliciviridae/virologia , Primers do DNA/química , Gastroenterite/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sapovirus/genética , Proteínas Estruturais Virais/genética , Sequência de Bases , Infecções por Caliciviridae/diagnóstico , Primers do DNA/genética , Fezes/virologia , Gastroenterite/diagnóstico , Expressão Gênica , Genótipo , Humanos , Tipagem Molecular/métodos , Filogenia , Sapovirus/classificação , Sapovirus/isolamento & purificação , Alinhamento de Sequência
8.
Clin Biochem ; 84: 73-78, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32592724

RESUMO

OBJECTIVES: A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) emerged in late 2019, causing an outbreak of pneumonia [coronavirus disease 2019 (COVID-19)] globally. Although the use of ready-made reaction mixes can enable more rapid PCR-based diagnosis of COVID-19, the need to transport and store these mixes at low temperatures presents challenges to already overburdened logistics networks. METHODS: Here, we present an optimized freeze-drying procedure that allows SARS-CoV-2 PCR mixes to be transported and stored at ambient temperatures, without loss of activity. Additive-supplemented PCR mixes were freeze-dried. The residual moisture of the freeze-dried PCR mixes was measured by Karl-Fischer titration. RESULTS: We found that the freeze-dried PCR mixes with ~1.2% residual moisture are optimal for storage, transport, and reconstitution. The sensitivity, specificity, and repeatability of the freeze-dried reagents were similar to those of freshly prepared, wet reagents. The freeze-dried mixes retained activity at room temperature (18 ~ 25 °C) for 28 days, and for 14 and 10 days when stored at 37 °C and 56 °C, respectively. CONCLUSION: The uptake of this approach will ease logistical challenges faced by transport networks and make more cold storage space available at diagnosis and hospital laboratories.


Assuntos
Betacoronavirus/genética , Técnicas de Laboratório Clínico/métodos , Infecções por Coronavirus/diagnóstico , Primers do DNA/química , DNA Viral/análise , Pneumonia Viral/diagnóstico , Reação em Cadeia da Polimerase/métodos , Betacoronavirus/isolamento & purificação , Infecções por Coronavirus/virologia , DNA Viral/genética , Liofilização , Humanos , Pandemias , Pneumonia Viral/virologia , Temperatura
9.
Nucleic Acids Res ; 48(12): e70, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32427335

RESUMO

Life emerging in an RNA world is expected to propagate RNA as hereditary information, requiring some form of primitive replication without enzymes. Non-enzymatic template-directed RNA primer extension is a model of the copying step in this posited form of replication. The sequence space accessed by primer extension dictates potential pathways to self-replication and, eventually, ribozymes. Which sequences can be accessed? What is the fidelity of the reaction? Does the recently illuminated mechanism of primer extension affect the distribution of sequences that can be copied? How do sequence features respond to experimental conditions and prebiotically relevant contexts? To help answer these and related questions, we here introduce a deep-sequencing methodology for studying RNA primer extension. We have designed and vetted special RNA constructs for this purpose, honed a protocol for sample preparation and developed custom software that analyzes sequencing data. We apply this new methodology to proof-of-concept controls, and demonstrate that it works as expected and reports on key features of the sequences accessed by primer extension.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala/métodos , RNA-Seq/métodos , Software , Primers do DNA/química , Primers do DNA/genética , Origem da Vida , RNA/química , RNA/genética
10.
Analyst ; 145(2): 440-444, 2020 Jan 21.
Artigo em Inglês | MEDLINE | ID: mdl-31793929

RESUMO

Polymerase chain reaction (PCR) and isothermal amplification methods such as LAMP and RPA are widely used for genetic detection. However, there are some shortcomings of these methods such as dependence on thermocycler instruments for PCR, complexity of primer design, the possibility for nonspecific amplification in LAMP and complexity of components in RPA. We develop a novel isothermal DNA detection system named Recombinase Assisted Loop-mediated Amplification (RALA). Recombinase from Thermus thermophilus (TthRecA) was used to open target double-stranded DNA to initiate loop-mediated amplification under isothermal conditions, which simplified the primer design and circumvented pre-denaturation. A FRET sensor named ProofMan and a proofreading enzyme Pfu were introduced to produce fluorescence signals by cleaving the sensor from the 3' end. Consequently, sequence-specific detection based on the RALA system was achieved, and even a single nucleotide polymorphism (SNP) could be identified. By introducing additional loop primers, the fast RALA version can amplify 102 DNA targets in 30 minutes. In addition to high sensitivity and specificity, the flexibility of choosing different reporting sensors makes this method versatile in either quantitative or qualitative DNA detection.


Assuntos
DNA/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Recombinases/química , DNA/genética , Primers do DNA/química , Transferência Ressonante de Energia de Fluorescência , Polimorfismo de Nucleotídeo Único , Thermus thermophilus/enzimologia
11.
Parasitol Res ; 119(1): 345-349, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31776667

RESUMO

Leishmaniasis is a parasitic disease of medical importance widely distributed around the world. Several methods are available for diagnosis but molecular approaches are highly recommended. To improve the sensitivity of an existing hsp20 gene based-PCR protocol to detect Leishmania parasites, primers were redesigned to amplify a shorter fragment using a new PCR variant (PCR-hsp20S). In this study, we aimed at characterizing the performance of the new method on cutaneous clinical samples and compare it with the former PCR-hsp20. The analytical sensitivity of the PCR-hsp20S was evaluated using DNA dilutions (100-0.1 pg) from Leishmania donovani and resulted in the detection of 10 fg of parasitic DNA, the equivalent to 0.05 parasite genome. For the diagnostic evaluation, a panel of 127 human clinical samples was used to calculate the parameters of sensitivity, specificity, accuracy, and positive and negative predictive values of the PCR-hsp20S. Diagnostic sensitivity was 94% (CI, 89.1-99.7%) and the specificity of 100% (CI, 98.6-100%). The same panel was also evaluated with the PCR-hsp20 to calculate the agreement between both molecular assays and to compare their performances. While both hsp20-based PCRs showed a good agreement coefficient (kappa index = 0.6), the performance of the novel variant, PCR-hsp20S, was significantly higher in terms of sensitivity (P = 0.0001) allowing the accurate detection of a higher number of Leishmania-positive clinical samples. We endorse the use of the PCR-hsp20S over the former protocol for the detection of Leishmania parasites from cutaneous clinical samples. In addition, as an improved sensitivity was achieved with the new method merely through the amplification of a shorter gene fragment, this investigation constitutes an experimental proof of this concept.


Assuntos
Proteínas de Choque Térmico HSP20/genética , Leishmania donovani/isolamento & purificação , Leishmaniose/parasitologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase/métodos , Animais , Primers do DNA/química , DNA de Protozoário/genética , Humanos , Leishmania donovani/genética , Proteínas de Protozoários/genética , Sensibilidade e Especificidade , Pele/parasitologia
12.
Eur J Ophthalmol ; 30(5): 901-907, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31232112

RESUMO

PURPOSE: To investigate vitamin D receptor polymorphisms in ocular surface squamous cell neoplasm and to evaluate the relationship between the identified polymorphisms and susceptibility to ocular surface squamous cell neoplasm and the clinical course. MATERIALS AND METHODS: A totala of 70 patients with ocular surface squamous cell neoplasm (study group) and 75 healthy age and gender-matched individuals (control group) were included in the study. Vitamin D receptor FokI and BsmI polymorphisms were examined. The relationships between histopathological diagnosis, recurrence rates, tumor stage, and identified polymorphisms were investigated. RESULTS: Histopathologically, 43 of the cases were squamous cell carcinoma and 27 of the cases were conjunctival intraepithelial neoplasia. The frequency of FokI (FF, Ff, ff) and BsmI (BB, Bb, bb) polymorphism genotype of vitamin D receptor gene were similar in the groups. The frequency of polymorphism (heterozygous or homozygous) for BsmI (Bb and bb) was significantly higher (p = 0.046) in the study group, while no difference was found between the groups in terms of polymorphic carriers (heterozygous or homozygous) for FokI. There was no correlation between tumor stage, recurrence-polymorphism frequency, and patient age-polymorphism frequency. CONCLUSION: It is known that active vitamin D inhibits the growth of cancer cells by binding to vitamin D receptor with regulation of genes responsible for cell proliferation. The presence of BsmI polymorphism in vitamin D receptor, in particular bb genotype and b allele, appears to be associated with the susceptibility of ocular surface squamous cell neoplasm. BsmI gene polymorphisms of vitamin D receptor might play an effective role in the formation, progression, and in the course of ocular surface squamous cell neoplasm.


Assuntos
Neoplasias da Túnica Conjuntiva/genética , Neoplasias de Células Escamosas/genética , Polimorfismo de Nucleotídeo Único , Receptores de Calcitriol/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias da Túnica Conjuntiva/patologia , Primers do DNA/química , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Feminino , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia , Neoplasias de Células Escamosas/patologia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único/genética , Estudos Prospectivos , Adulto Jovem
13.
Sci Rep ; 9(1): 17707, 2019 11 27.
Artigo em Inglês | MEDLINE | ID: mdl-31776407

RESUMO

Genome walking (GW) refers to the capture and sequencing of unknown regions in a long DNA molecule that are adjacent to a region with a known sequence. A novel PCR-based method, palindromic sequence-targeted PCR (PST-PCR), was developed. PST-PCR is based on a distinctive design of walking primers and special thermal cycling conditions. The walking primers (PST primers) match palindromic sequences (PST sites) that are randomly distributed in natural DNA. The PST primers have palindromic sequences at their 3'-ends. Upstream of the palindromes there is a degenerate sequence (8-12 nucleotides long); defined adapters are present at the 5'-termini. The thermal cycling profile has a linear amplification phase and an exponential amplification phase differing in annealing temperature. Changing the annealing temperature to switch the amplification phases at a defined cycle controls the balance between sensitivity and specificity. In contrast to traditional genome walking methods, PST-PCR is rapid (two to three hours to produce GW fragments) as it uses only one or two PCR rounds. Using PST-PCR, previously unknown regions (the promoter and intron 1) of the VRN1 gene of Timothy-grass (Phleum pratense L.) were captured for sequencing. In our experience, PST-PCR had higher throughput and greater convenience in comparison to other GW methods.


Assuntos
Genoma de Planta , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Sequências Repetidas Invertidas , Reação em Cadeia da Polimerase/métodos , Análise de Sequência de DNA/métodos , Primers do DNA/química , Primers do DNA/genética , Sequenciamento de Nucleotídeos em Larga Escala/normas , Íntrons , Proteínas de Plantas/genética , Poaceae/genética , Reação em Cadeia da Polimerase/normas , Regiões Promotoras Genéticas , Proteínas Repressoras/genética , Análise de Sequência de DNA/normas , Temperatura
14.
Invest Ophthalmol Vis Sci ; 60(14): 4904-4914, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31770435

RESUMO

Purpose: Uveal melanoma is a common primary intraocular malignancy accompanied by high mortality. Previous evidence has highlighted the implication of microRNAs (miRNAs) and long noncoding RNAs (lncRNAs) in uveal melanoma. Accordingly, we further uncovered the possible role of lncRNA plasmacytoma variant translocation 1 gene (PVT1) and microRNA-17-3p (miR-17-3p) in uveal melanoma. Methods: A series of experiments were performed to examine the relationship among lncRNA PVT1, miR-17-3p, and murine double minute clone 2 oncoprotein (MDM2). Afterward, gain- and loss-of-function approaches were used with uveal melanoma cells to verify the role of lncRNA PVT1, miR-17-3p, and MDM2 in the tumorigenesis and development of uveal melanoma. Results: Highly expressed lncRNA PVT1 and MDM2, yet lowly expressed miR-17-3p, were identified in ocular uveal melanoma tissues versus normal adjacent tissues. Then, dual luciferase reporter gene assay, RNA binding protein immunoprecipitation, and RNA pull-down assays showed that lncRNA PVT1 specifically bound to miR-17-3p, and that MDM2 was a target gene of miR-17-3p. Gain- and loss-of-function studies elucidated that silencing of lncRNA PVT1 or overexpression of miR-17-3p resulted in decreased MDM2 expression and increased transcriptional activity of p53, in addition to inhibiting uveal melanoma cell proliferation, migration, and invasion, yet promoted cell apoptosis in vitro. In addition, lncRNA PVT1 silencing or miR-17-3p overexpression was noted to inhibit tumor growth in vivo. Conclusions: Downregulation of lncRNA PVT1 could potentially promote miR-17-3p expression to suppress tumorigenesis and development of uveal melanoma by activating the p53 signaling pathway through binding to MDM2.


Assuntos
Inativação Gênica/fisiologia , Melanoma/prevenção & controle , MicroRNAs/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , RNA Longo não Codificante/genética , Neoplasias Uveais/prevenção & controle , Animais , Western Blotting , Movimento Celular/fisiologia , Sobrevivência Celular , Primers do DNA/química , Técnica Indireta de Fluorescência para Anticorpo , Genes Reporter , Hibridização in Situ Fluorescente , Masculino , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais , Ativação Transcricional , Transplante Heterólogo , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Neoplasias Uveais/metabolismo
15.
Parasitol Res ; 118(12): 3469-3478, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31691003

RESUMO

Blastocystis is a prevalent parasite that has a wide distribution. In order to design HRM real-time PCR, primers were selected from SSU rRNA gene to amplify specific fragment with different melting temperatures for each subtype of Blastocystis. Subsequently, HRM real-time PCR was performed and melting curve analysis was done by Rotor-Gene Q software. The results of HRM real-time PCR was then compared with sequence results of "barcoding region" of SSU rRNA gene of Blastocystis. To evaluate sensitivity of test, 10-fold serial dilutions of the parasite were prepared from ~ 106 to 1 parasite per mL of stool sample and were investigated by HRM real-time PCR. In order to determine specificity of method, HRM real-time PCR was done for some microorganisms and Blastocystis-negative stool samples. In silico analysis showed that all seventeen subtypes of Blastocystis were distinguish. In vitro analysis revealed that the test discriminated subtypes with specific melting temperatures.


Assuntos
Infecções por Blastocystis/parasitologia , Blastocystis/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/métodos , Blastocystis/classificação , Blastocystis/genética , Infecções por Blastocystis/diagnóstico , Primers do DNA/química , Primers do DNA/genética , DNA de Protozoário/química , DNA de Protozoário/genética , Testes Diagnósticos de Rotina , Fezes/parasitologia , Humanos , Sensibilidade e Especificidade
16.
Sci Rep ; 9(1): 15789, 2019 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-31673037

RESUMO

The development and application of next-generation sequencing (NGS) have enabled comprehensive analyses of the microbial community through extensive parallel sequencing. Current analyses of the eukaryotic microbial community are primarily based on polymerase chain reaction amplification of 18S rRNA gene (rDNA) fragments. We found that widely-used 18S rDNA primers can amplify numerous stretches of the bacterial 16S rRNA gene, preventing the high-throughput detection of rare eukaryotic species, particularly in bacteria-rich samples such as faecal material. In this study, we employed in silico and NGS-based analyses of faecal samples to evaluated the existing primers targeting eukaryotic 18S and 28S rDNA in terms of avoiding bacterial read contamination and improving taxonomic coverage for eukaryotes, with a particular emphasis on parasite taxa. Our findings revealed that newly selected primer sets could achieve these objectives, representing an alternative strategy for NGS.


Assuntos
Primers do DNA , Eucariotos , Sequenciamento de Nucleotídeos em Larga Escala , Parasitos , Reação em Cadeia da Polimerase , RNA Ribossômico 16S/genética , RNA Ribossômico 18S/genética , Animais , Primers do DNA/química , Primers do DNA/genética , Eucariotos/classificação , Eucariotos/genética , Parasitos/classificação , Parasitos/genética
17.
Molecules ; 24(19)2019 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-31591283

RESUMO

Aptamers are small oligonucleotides that are capable of binding specifically to a target, with impressive potential for analysis, diagnostics, and therapeutics applications. Aptamers are isolated from large nucleic acid combinatorial libraries using an iterative selection process called SELEX (Systematic Evolution of Ligands by EXponential enrichment). Since being implemented 30 years ago, the SELEX protocol has undergone many modifications and improvements, but it remains a laborious, time-consuming, and costly method, and the results are not always successful. Each step in the aptamer selection protocol can influence its results. This review discusses key technical points of the SELEX procedure and their influence on the outcome of aptamer selection.


Assuntos
Aptâmeros de Nucleotídeos/química , Primers do DNA/química , Técnica de Seleção de Aptâmeros/métodos , Aptâmeros de Nucleotídeos/síntese química , Primers do DNA/síntese química , DNA de Cadeia Simples/isolamento & purificação , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Técnicas de Amplificação de Ácido Nucleico , Ácidos Nucleicos/química , Reação em Cadeia da Polimerase
18.
Biotechniques ; 67(6): 271-275, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31631692

RESUMO

This study evaluated the effectiveness of thermocycler temperature control, considering the influence of other determinant factors for the optimization of PCR. The reduction in the number of repeated PCR tests, applied in the diagnosis and prognosis of chronic myeloid leukemia at the National Cancer Institute in Brazil, was used as a measure of effectiveness. This indicator was evaluated using samples obtained before and after the temperature control in the wells of the thermocyclers. There was a reduction of 18.9% in the number of repeated exams in the second sample. A structured interview with laboratory staff indicated that there was no change in the other determinant factors.


Assuntos
Reação em Cadeia da Polimerase/métodos , Brasil , Primers do DNA/química , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Temperatura
19.
Mol Biotechnol ; 61(12): 938-944, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31641996

RESUMO

The Hot Start polymerase chain reaction (Hot Start PCR) is designed to reduce off-target amplification by blocking DNA polymerase extension at room temperature until the desired temperature is reached. In this study, we investigated a new method of Hot Start PCR that uses a modified Escherichia coli Exonuclease III (EcoExoIIIM) by substituting residues in the DNA-binding pocket and catalytic center. The results showed that PCR amplification yield and specificity were significantly promoted by the addition of EcoExoIIIM. We hypothesize that non-specific binding of primers at room temperature is prevented by binding of the primed template by EcoExoIIIM, which is then released from the DNA by heat denaturation before the first PCR cycle. Through this mechanism, PCR would be enhanced by reducing off-target extension at room temperature.


Assuntos
Escherichia coli/enzimologia , Escherichia coli/genética , Exodesoxirribonucleases/genética , Reação em Cadeia da Polimerase/métodos , Primers do DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases/química , Exodesoxirribonucleases/metabolismo , Temperatura Alta , Mutação , Temperatura
20.
Exp Parasitol ; 206: 107754, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31473211

RESUMO

Dermatophagoides farinae is an important source of indoor allergens that shows strong tolerance to external temperatures. However, the regularity and mechanism of tolerance are still unclear. Based on our previous RNA-seq and annotation of D. farinae under temperature stress, it is planned to identify differentially expressed genes (DEGs) involved in the temperature stress response by quantitative real-time PCR (qRT-PCR). However, the lack of reference genes directly limited the detection and confirmation of DEGs. Accordingly, in this study, we have selected six candidates as reference genes in D. farinae: 60S RP L11, 60S RP L21, α tubulin, GAPDH, Der f Mal f 6, and calreticulin, and evaluated their expression stabilities as affected by heat and cold stresses, using geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder methods. Then the expression level of 15 DEGs were detected and verified. geNorm analysis showed that α tubulin and calreticulin were the most stable reference genes under heat stress and cold stress of D. farinae. Similar evaluation results were obtained by NormFinder and BestKeeper, in which 60S RP L21 and α tubulin were the most stable reference genes. By comparative ΔCt method and a comprehensive evaluation of RefFinder, α tubulin was identified as the most ideal reference gene of D. farinae under heat and cold stresses. Furthermore, qRT-PCR detection results of 15 DEGs were almost identical to the RNA-seq results, indicating that α tubulin is stable as a reference gene. This study provided technical support for DEGs expression studies in D. farinae using qRT-PCR.


Assuntos
Calreticulina/genética , Dermatophagoides farinae/genética , Temperatura , Tubulina (Proteína)/genética , Animais , Antígenos de Dermatophagoides/genética , Primers do DNA/química , Dermatophagoides farinae/fisiologia , Feminino , Amplificação de Genes , Perfilação da Expressão Gênica , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Anotação de Sequência Molecular , RNA/química , RNA/genética , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Proteínas Ribossômicas/genética , Análise de Sequência de RNA , Transcriptoma/genética , Temperatura de Transição , Sequenciamento Completo do Exoma
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