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1.
World J Microbiol Biotechnol ; 35(11): 176, 2019 Oct 31.
Artigo em Inglês | MEDLINE | ID: mdl-31673867

RESUMO

The aim of this study was to clarify effects of soil and climatic conditions on community structure of sweet potato bacterial endophytes by applying locked nucleic acid oligonucleotide-PCR clamping technique and metagenomic analysis. For this purpose, the soil samples in three locations were transferred each other and sweet potato nursery plants from the same farm were cultivated for ca. 3 months. After removal of plastid, mitochondria and undefined sequences, the averaged numbers of retained sequences and operational taxonomic units per sample were 20,891 and 846, respectively. Proteobacteria (85.0%), Bacteroidetes (6.6%) and Actinobacteria (6.3%) were the three most dominant phyla, accounting for 97.9% of the reads, and γ-Proteobacteria (66.3%) being the most abundant. Top 10 genera represented 81.2% of the overall reads in which Pseudomonas (31.9-45.0%) being the most predominant. The overall endophytic bacterial communities were similar among the samples which indicated that the soil and the climatic conditions did not considerably affect the entire endophytic community. The original endophytic bacterial community might be kept during the cultivation period.


Assuntos
Bactérias/classificação , Bactérias/metabolismo , Clima , Endófitos/classificação , Ipomoea batatas/microbiologia , Metagenoma , Microbiota , Solo/química , Bactérias/genética , Sequência de Bases , Biodiversidade , DNA Bacteriano/análise , DNA Mitocondrial/análise , Endófitos/genética , Microbiota/genética , Filogenia , RNA Ribossômico 16S/genética , Microbiologia do Solo
2.
Lett Appl Microbiol ; 69(6): 411-416, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31563155

RESUMO

Plesiomonas shigelloides is a common pathogen of aquatic animals and can pose a certain hazard to aquaculture. Here, we aimed to develop a loop-mediated isothermal amplification (LAMP) method for the visual detection of P. shigelloides to aid the diagnosis of infections caused by this pathogen in aquatic animals. We used LAMP to amplify P. shigelloides DNA and combined it with calcein or nucleic acid dipstick assay (NADA) to visualize the amplified products. The optimal LAMP amplification temperature was 64°C, and the reaction lasted for 50 min. The limit of detection of recombinant plasmids containing the target gene using the LAMP method was 2·0 × 102 copies per µl, which is ten times higher than that using conventional polymerase chain reaction (PCR). LAMP products could be visualized without agarose gel electrophoresis. We tested 85 fish specimens using the established LAMP method and conventional PCR. The detection rate was 42·4% using the LAMP method and 34·1% using conventional PCR. Based on our results, the LAMP method combined with calcein or NADA is a rapid, specific, sensitive and accurate method for visual detection of fish-derived P. shigelloides and can be used for the laboratory diagnosis of infections caused by it. SIGNIFICANCE AND IMPACT OF THE STUDY: The combination of loop-mediated isothermal amplification (LAMP) and calcein and nucleic acid dipstick assay (NADA) provided a rapid, specific and sensitive method for detecting Plesiomonas shigelloides, which is an important pathogen that causes diseases in aquatic animals worldwide. In the present study, the LAMP method showed a higher detection rate than conventional PCR for P. shigelloides using templates from 85 fish specimens. Thus, the LAMP method could be a reliable and convenient tool for diagnosing diseases in aquatic animals in the laboratory.


Assuntos
DNA Bacteriano/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Plesiomonas/isolamento & purificação , Animais , Aquicultura , Peixes/microbiologia , Plesiomonas/genética , Reação em Cadeia da Polimerase , Sensibilidade e Especificidade
3.
Cell Host Microbe ; 26(3): 325-335.e5, 2019 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-31492655

RESUMO

Bacteriophages are abundant within the human gastrointestinal tract, yet their interactions with gut bacteria remain poorly understood, particularly with respect to CRISPR-Cas immunity. Here, we show that the type I-C CRISPR-Cas system in the prevalent gut Actinobacterium Eggerthella lenta is transcribed and sufficient for specific targeting of foreign and chromosomal DNA. Comparative analyses of E. lenta CRISPR-Cas systems across (meta)genomes revealed 2 distinct clades according to cas sequence similarity and spacer content. We assembled a human virome database (HuVirDB), encompassing 1,831 samples enriched for viral DNA, to identify protospacers. This revealed matches for a majority of spacers, a marked increase over other databases, and uncovered "hyper-targeted" phage sequences containing multiple protospacers targeted by several E. lenta strains. Finally, we determined the positional mismatch tolerance of observed spacer-protospacer pairs. This work emphasizes the utility of merging computational and experimental approaches for determining the function and targets of CRISPR-Cas systems.


Assuntos
Bactérias/virologia , Bacteriófagos/genética , Sistemas CRISPR-Cas , Trato Gastrointestinal/microbiologia , Actinobacteria/virologia , Bactérias/genética , Sequência de Bases , DNA Bacteriano/análise , DNA Viral/análise , Bases de Dados Genéticas , Genoma Bacteriano , Genoma Viral , Humanos , Metagenômica , Microbiota/genética , Análise de Sequência de DNA
4.
Anal Chim Acta ; 1080: 162-169, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31409466

RESUMO

Driven by a bright prospect for rapid, portable and cost-effective point-of-care testing, an assembled Pasteur pipette device to integrate nucleic acid extraction, amplification and detection was developed to detect B. cereus in a sample-to-answer format. Denaturation Bubble-mediated Strand Exchange Amplification (SEA) was chosen to perform isothermal amplification because it requires only a pair of primers and one Bst DNA polymerase. The established SEA can detect as low as 1.0 × 10-13 M genomic DNA of B. cereus, which was comparable with the previously reported method for B. cereus detection. The assembled Pasteur pipette allows sample-to-answer diagnostic in a simple, low-cost, portable, and disposable format. The inherent function of Pasteur pipette enables direct liquid handling without the need of extra pipettes, syringes or pumps. Visual readout was achieved by using a pH sensitive dye, further simplifying result judgment process. The detection limit for B. cereus is 1.0 × 104 CFU/mL in pure cultures, while the detection limit in artificially contaminated milk is 1.0 × 105 CFU/mL without enrichment and 1.0 × 100 CFU/mL following 12 h enrichment. Considering that typical cell counts in food samples associated to food poisoning are 1.0 × 105 to 1.0 × 108 CFU per gram/milliliter B. cereus, our Pasteur pipette is enough sensitive for answer-to-sample diagnosis of B. cereus even directly from foods without enrichment. The whole diagnostic procedure could be completed within 50 min, dramatically decreasing the detection time. In a word, the assembled Pasteur pipette device, combined with a homemade metal bath, possesses great potential for sample-to-answer application in resource-limited settings.


Assuntos
Bacillus cereus/isolamento & purificação , Carga Bacteriana/métodos , Colorimetria/métodos , DNA Bacteriano/análise , Animais , Carga Bacteriana/instrumentação , Proteínas de Bactérias/química , Colorimetria/instrumentação , Corantes/química , DNA Polimerase Dirigida por DNA/química , Contaminação de Alimentos/análise , Geobacillus stearothermophilus/enzimologia , Limite de Detecção , Leite/microbiologia , Técnicas de Amplificação de Ácido Nucleico/métodos , Papel
5.
Anal Chim Acta ; 1080: 75-83, 2019 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-31409477

RESUMO

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), remains the top fatal infection continuing to threat public health, and the present detection method for MTB is facing great challenges with the global TB burden. In response to this issue, a novel electrochemical DNA biosensor was developed for detecting the IS6110 fragment within MTB. For the first time, the nanohybrid of gold nanoparticles decorated fullerene nanoparticles/nitrogen-doped graphene nanosheet (Au-nano-C60/NGS) directly served as a new signal tag to generate signal response without additional redox molecules and subsequently labeled with signal probes (SPs) to form tracer label to achieve signal amplification. Additionally, a biotin-avidin system was introduced to immobilize abundant capture probes (CPs), further improving the sensitivity of the proposed biosensor. After a typical sandwich hybridization, the proposed electrochemical DNA biosensor was incubated with tetraoctylammonium bromide (TOAB), which was used as a booster to induce the intrinsic redox activity of the tracer label, resulting in a discriminating current response. The proposed electrochemical DNA biosensor shows a broad linear range for MTB determination from 10 fM to 10 nM with a low limit of detection (LOD) of 3 fM. In addition, this proposed biosensor not only distinguishes mismatched DNA sequence, but also differentiates MTB from other pathogenic agents. More importantly, it has been preliminarily applied in clinical detection and displayed excellent ability to identify the PCR products of clinical samples. There is great potential for this developed method to be used in early diagnosis and monitor of TB.


Assuntos
DNA Bacteriano/análise , Fulerenos/química , Grafite/química , Nanopartículas Metálicas/química , Mycobacterium tuberculosis/isolamento & purificação , Nitrogênio/química , Técnicas Biossensoriais/métodos , Candida albicans/genética , Sondas de DNA/genética , DNA Bacteriano/genética , Técnicas Eletroquímicas/métodos , Ouro/química , Limite de Detecção , Mycobacterium tuberculosis/genética , Nanocompostos/química , Hibridização de Ácido Nucleico , Reprodutibilidade dos Testes , Staphylococcus aureus/genética
6.
Niger J Clin Pract ; 22(8): 1083-1090, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31417051

RESUMO

Aims: The aim of this study was to provide epidemiological data about the presence of Salmonella spp. and Shigella spp. in raw milk samples collected from different animals. Methods: A total of 231 raw milk samples from 48 cows, 65 goats, 65 sheep, and 53 donkeys were studied. The ISO 6579:2002 and ISO 21567:2004 methods, antimicrobial susceptibility tests, and serotyping were performed. Species and subspecies discriminations were made via matrix-assisted laser desorption/ionization-time of flight mass spectrometry. After DNA isolation from all samples, Salmonella spp. and Shigella spp. were detected using real-time polymerase chain reaction (PCR) kits. Results: Five samples (2.16%) showed positivity out of 231 raw milk samples for Salmonella spp., and 2 (0.87%) samples were detected to be positive by multiplex real-time PCR design. Conclusion: We found that raw milk samples were not free of Salmonella spp. and Shigella spp. and need to be tested routinely to avoid public health problems. Rapid and reliable real-time PCR method can be developed and used for this purposes instead of slow bacterial culture processes.


Assuntos
DNA Bacteriano/análise , Contaminação de Alimentos/análise , Leite/microbiologia , Reação em Cadeia da Polimerase/métodos , Salmonella/genética , Salmonella/isolamento & purificação , Shigella/genética , Shigella/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Animais , Bovinos , Equidae , Feminino , Cabras , Humanos , Salmonella/classificação , Sensibilidade e Especificidade , Ovinos , Shigella/classificação
7.
Arq Gastroenterol ; 56(2): 141-145, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31460576

RESUMO

BACKGROUND: Colorectal cancer is one of the most commonly diagnosed cancers around the world. One of the factors involved in the development of colorectal cancer is the changes in the normal flora of the intestine. OBJECTIVE: In this study, the mean copy number of Enterococcus faecalis in people with polyps and people with colorectal cancer has been evaluated in comparison with healthy controls. METHODS: In this study, 25 patients with colorectal cancer and 28 patients with intestinal polyps were selected and stool specimens were taken. In addition, 24 healthy individuals were selected as control group. Extraction of bacterial DNA from the stool sample were performed. The molecular methods of PCR for confirmation of standard strain and absolute Real Time PCR (qRT-PCR) method were used to evaluate the number of Enterococcus faecalis in the studied groups. RESULTS: The results of this study indicate that the mean copy number of Enterococcus faecalis in patients with colorectal cancer was 11.2x109 per gram of stool, and in patients with polyps was 9.4x108 per gram of stool. In healthy people, this number was 9x108 per gram of stool. There was a significant difference between the implicit copy numbers in the three groups. (P<0.05). CONCLUSION: Enterococcus faecalis in faecal flora of people with colorectal cancer was significantly higher than those with polyps and healthy people. This could potentially signify the ability of this bacterium to induce colorectal cancer. More studies are needed to prove this theory.


Assuntos
Pólipos do Colo/microbiologia , Neoplasias Colorretais/microbiologia , Enterococcus faecalis/isolamento & purificação , Fezes/microbiologia , Idoso , Estudos de Casos e Controles , DNA Bacteriano/análise , Enterococcus faecalis/genética , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase em Tempo Real
8.
J Vet Diagn Invest ; 31(5): 792-794, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31423914

RESUMO

We report herein the use of crude extracts obtained from samples of Taylorella equigenitalis-infected horses for the purpose of multi-locus sequence typing (MLST). Samples (n = 36) were collected from horses in South Africa from 1996 to 2017: 34 from genital swabs (stored at -20°C for 2-3 y) and 2 from cryopreserved raw semen aliquots (stored at -70°C for 18 y) prior to assay. The MLST assay showed a single sequence type (ST), designated ST4, that supported a point introduction and thus a common source for the South African outbreak of contagious equine metritis.


Assuntos
Infecções por Bactérias Gram-Negativas/veterinária , Doenças dos Cavalos/diagnóstico , Tipagem de Sequências Multilocus/veterinária , Infecções do Sistema Genital/veterinária , Sêmen/microbiologia , Taylorella equigenitalis/isolamento & purificação , Animais , DNA Bacteriano/análise , Feminino , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/microbiologia , Doenças dos Cavalos/microbiologia , Cavalos , Masculino , Infecções do Sistema Genital/microbiologia , África do Sul
9.
IET Nanobiotechnol ; 13(6): 602-608, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31432793

RESUMO

A single pot, green method for platinum nanoparticles (Pt NP) production was devised with gum ghatti (Anogeissus latifolia). Analytical tools: ultraviolet-visible (UV-vis), dynamic light scattering, zeta potential, transmission electron microscope, X-ray diffraction (XRD), and Fourier transform infrared spectroscopy were employed. Wide continuous UV-vis absorption and black solution colouration proved Pt NP formation. Face-centred cubic crystalline structure of NP was evidenced from XRD. NPs formed were nearly spherical with a mean particle size of 3 nm. NP demonstrated a myriad of properties including catalytic, peroxidase, polymerase chain reaction (PCR) enhancing and antioxidant activities. Catalytic action of NP was probed via NaBH4 reduction of arsenazo-III dye. NP displayed considerable peroxidase activity via catalysis of 3, 3', 5, 5'-tetramethylbenzidine oxidation by H2O2. NP showed exceptional stability towards varying pH (3-11), temperature (25-100°C), salt concentration (0-100 mM) and storage time duration (0-12 months). In comparison with horse radish peroxidase, its applicability as an artificial peroxidase is advantageous. NP caused a two-fold enhancement in PCR yield at 0.4 nM. Also showed significant 1', 1' diphenyl picryl-hydrazyle scavenging (80.1%) at 15 µg/mL. Author envisages that the biogenic Pt NP can be used in a range of biological and environmental applications.


Assuntos
Química Verde/métodos , Nanopartículas Metálicas/química , Gomas Vegetais/química , Platina/farmacologia , Antioxidantes/química , Antioxidantes/farmacologia , Catálise/efeitos dos fármacos , DNA Bacteriano/análise , DNA Bacteriano/efeitos dos fármacos , DNA Fúngico/análise , DNA Fúngico/efeitos dos fármacos , Peróxido de Hidrogênio/química , Peróxido de Hidrogênio/metabolismo , Teste de Materiais , Testes de Sensibilidade Microbiana , Oxirredução/efeitos dos fármacos , Peroxidases/efeitos dos fármacos , Peroxidases/metabolismo , Platina/química , Reação em Cadeia da Polimerase/métodos , Pseudomonas aeruginosa/genética
10.
Analyst ; 144(19): 5766-5774, 2019 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-31436781

RESUMO

We present a centrifugal microfluidic device which is combined with a solution-loading cartridge for fully automatic molecular diagnostics of foodborne pathogens. The proposed device could perform all processes of molecular diagnostics including bead-based DNA extraction, isothermal DNA amplification and colorimetric amplicon detection. In particular, the 3D-printed solution-loading cartridge was incorporated into the device to enhance the sample handling capacity and eliminate laborious steps such as pipetting for sample loading and sealing the top of the device. The cartridge could store four kinds of essential solutions (a sample solution, a washing solution, an elution solution, and a loop-mediated isothermal amplification (LAMP) cocktail) for pathogen detection, and the designated solutions were automatically released into the microdevice in a consecutive order by rotation. Since one unit of a device contains 20 reaction chambers, 18 kinds of pathogens plus two controls can be simultaneously detected in one test. As a proof-of-concept, we targeted four kinds of foodborne pathogens (Escherichia coli O157:H7, Salmonella typhimurium, Vibrio parahaemolyticus and Listeria monocytogenes) and successfully verified them, demonstrating that the centrifugal microdevice could be combined with a 3D printed solution-loading cartridge to achieve fully automated lab-on-a-chip-based molecular diagnostics. The entire process was completed in 65 min, and the limit of detection of the assay was 100 bacterial cells. The employment of the solution-loading cartridge successfully replaced the laborious and error-prone manual loading processes, which realized true automation of molecular diagnostics. This device could have promise in the fields of lab-on-a-chip and point-of-care molecular diagnostics.


Assuntos
DNA Bacteriano/análise , Doenças Transmitidas por Alimentos/microbiologia , Dispositivos Lab-On-A-Chip , Técnicas de Diagnóstico Molecular/métodos , Bactérias/genética , Bactérias/isolamento & purificação , Técnicas Biossensoriais/instrumentação , Técnicas Biossensoriais/métodos , Centrifugação , Colorimetria , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/diagnóstico , Técnicas de Diagnóstico Molecular/instrumentação , Técnicas de Amplificação de Ácido Nucleico/métodos , Impressão Tridimensional , Estudo de Prova de Conceito
11.
Nature ; 572(7769): 329-334, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31367035

RESUMO

We sought to determine whether pre-eclampsia, spontaneous preterm birth or the delivery of infants who are small for gestational age were associated with the presence of bacterial DNA in the human placenta. Here we show that there was no evidence for the presence of bacteria in the large majority of placental samples, from both complicated and uncomplicated pregnancies. Almost all signals were related either to the acquisition of bacteria during labour and delivery, or to contamination of laboratory reagents with bacterial DNA. The exception was Streptococcus agalactiae (group B Streptococcus), for which non-contaminant signals were detected in approximately 5% of samples collected before the onset of labour. We conclude that bacterial infection of the placenta is not a common cause of adverse pregnancy outcome and that the human placenta does not have a microbiome, but it does represent a potential site of perinatal acquisition of S. agalactiae, a major cause of neonatal sepsis.


Assuntos
Parto Obstétrico , Complicações do Trabalho de Parto/microbiologia , Placenta/microbiologia , Complicações Infecciosas na Gravidez/microbiologia , Sepse/congênito , Sepse/microbiologia , Streptococcus agalactiae/isolamento & purificação , Streptococcus agalactiae/patogenicidade , Biópsia , Estudos de Coortes , Contaminação por DNA , DNA Bacteriano/análise , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Feminino , Humanos , Recém-Nascido , Masculino , Metagenômica , Gravidez , Resultado da Gravidez , RNA Ribossômico 16S/análise , RNA Ribossômico 16S/genética , Reprodutibilidade dos Testes , Análise de Sequência de DNA
12.
Eur J Clin Microbiol Infect Dis ; 38(10): 1891-1899, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31367996

RESUMO

There is increasing evidence indicating a role for Fusobacterium nucleatum (F. nucleatum) in colorectal cancer (CRC) development and prognosis. This study evaluated F. nucleatum as a prognostic biomarker, by assessing its association with post-diagnosis survival from CRC. From September 2008 to April 2012 CRC patients (n = 190) were recruited from three hospitals within the Czech Republic. F. nucleatum DNA copies were measured in adjacent non-malignant and colorectal tumor tissues using quantitative real-time PCR. Cox Proportional Hazards (HR) models were applied to evaluate the association between F. nucleatum DNA and overall survival, adjusting for key confounders. Risk prediction modeling was conducted to evaluate the ability to predict survival based on F. nucleatum status. High, compared with low, levels of F. nucleatum in colorectal tumor tissues were associated with poorer overall survival (adjusted HR 1.68, 95% CI 1.02-2.77), which was slightly attenuated after additional adjustment for microsatellite instability status. However, inclusion of F. nucleatum in risk prediction models did not improve the ability to identify patients who died beyond known prognostic factors such as disease pathology staging. Although the increased presence of F. nucleatum was associated with poorer prognosis in CRC patients, this may have limited clinical relevance as a prognostic biomarker.


Assuntos
Biomarcadores/análise , Neoplasias Colorretais/patologia , DNA Bacteriano/análise , Infecções por Fusobacterium/microbiologia , Fusobacterium nucleatum/genética , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Neoplasias Colorretais/mortalidade , República Tcheca , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Reação em Cadeia da Polimerase em Tempo Real , Medição de Risco , Análise de Sobrevida
13.
N Z Vet J ; 67(6): 329-332, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31378159

RESUMO

Aims: To determine if presence of the Bacteroides fragilis toxin (bft) gene, a molecular marker of colonic carriage of entertoxigenic Bacteroides fragilis (ETBF) in humans, was associated with a finding of small intestinal adenocarcinomas (SIA) in sheep in New Zealand. Methods: Samples of jejunal tissue were collected from the site of tumours and from grossly normal adjacent tissue in 20 sheep, in different consignments, diagnosed with SIA based on gross examination of viscera following slaughter. Two jejunal samples were also collected from a control sheep in the same consignment that had no gross evidence of SIA. A PCR assay was used to detect the presence of the bft gene in the samples. Results: Of the sheep with SIA, the bft gene was amplified from one or both samples from 7/20 (35%) sheep, and in sheep that had no gross evidence of SIA the bft gene was amplified from at least one sample in 11/20 (55%) sheep (RR 0.61; 95% CI = 0.30-1.25; p = 0.34). Of 11 positive samples analysed, ETBF subtype bft-1 was detected in one, bft-2 was detected in 10, and none were bft-3. Conclusions and Clinical Relevance: There was a high prevalence of detection of the bft gene in both SIA-affected and non-affected sheep, but there was no apparent association between carriage of ETBF, evidenced by detection of the bft gene, and the presence of SIA. ETBF are increasingly implicated in the aetiology of human colorectal cancer, raising the possibility that sheep may provide a zoonotic reservoir of this potentially carcinogenic bacterium. Abbreviation: Bft: Bacteroides fragilis toxin; ETBF: Enterotoxigenic Bacteroides fragilis; SIA: Small intestinal adenocarcinoma.


Assuntos
Adenocarcinoma/veterinária , Toxinas Bacterianas/genética , Neoplasias Intestinais/veterinária , Metaloendopeptidases/genética , Doenças dos Ovinos/diagnóstico , Adenocarcinoma/diagnóstico , Adenocarcinoma/microbiologia , Animais , Toxinas Bacterianas/isolamento & purificação , DNA Bacteriano/análise , Genes Bacterianos , Neoplasias Intestinais/diagnóstico , Neoplasias Intestinais/microbiologia , Metaloendopeptidases/isolamento & purificação , Ovinos , Doenças dos Ovinos/microbiologia
14.
Analyst ; 144(18): 5413-5419, 2019 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-31364999

RESUMO

Herein, a chip imitating the desert beetle shell was presented for naked eye nucleic acid quantification. The hydrophobic photonic crystal substrate treated by ultraviolet local irradiation could effectively disperse the sample into hundreds of droplets for digital loop-mediated isothermal amplification (dLAMP). Pyrophosphate (PPI), a by-product of the LAMP reaction, combined with magnesium ions to form a poorly soluble precipitate. It could be fixed on a silica substrate due to complexation, resulting in the disappearance of the structural color of the photonic crystals. The number of points without structural color contains the information of the copy number of nucleic acids in the sample. This chip could achieve the naked eye quantitative detection of Salmonella DNA without fluorescence or other chromogenic reagents. Thus, the chip designed in this study can help the development of digital nucleic acid detection under limited resource settings (LRS) and can be suitable for POCT (point of care test) standards.


Assuntos
Materiais Biomiméticos/química , DNA Bacteriano/análise , Fluorcarbonetos/química , Silanos/química , Colorimetria/métodos , Difosfatos/química , Fluorcarbonetos/efeitos da radiação , Interações Hidrofóbicas e Hidrofílicas , Compostos de Magnésio/química , Nanopartículas/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Estudo de Prova de Conceito , Salmonella/genética , Silanos/efeitos da radiação , Dióxido de Silício/química , Raios Ultravioleta
15.
Biosens Bioelectron ; 142: 111541, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31382097

RESUMO

Some of microorganisms are potential pathogens that can be infectious agents under some circumstances, and development of new detection methods of the pathogens is of high interest. In the present study, an Enterococcus faecalis (E. faecalis) DNA biosensor (ef-biosensor) was fabricated to quantify the bacterium genome. A specific E. faecalis DNA probe was selected from 16S rRNA sequence of E. faecalis and immobilized on a gold electrode surface in an optimized time to fabricate the ef-biosensor. The ef-biosensor detected a synthetic target of the probe with a detection limit of 3.3 amol L-1 and with a nice selectivity to resolve from one-, two- and three-base mismatched sequences. In addition, the bacterium genomic DNA was quantified with a detection limit of 7.1 × 10-9 ng mL-1 in a concentration range of 1.1 × 10-7 to 1.1 ng mL-1. The ef-biosensor had a long time stability, good fabrication reproducibility and good regeneration ability. The ef-biosensor was successfully applied for E. faecalis detection in human samples.


Assuntos
Técnicas Biossensoriais/métodos , Sondas de DNA/química , DNA Bacteriano/análise , Enterococcus faecalis/isolamento & purificação , Infecções por Bactérias Gram-Positivas/microbiologia , RNA Ribossômico 16S/química , Sequência de Bases , Sondas de DNA/genética , DNA Bacteriano/genética , Técnicas Eletroquímicas/métodos , Enterococcus faecalis/genética , Infecções por Bactérias Gram-Positivas/diagnóstico , Humanos , Limite de Detecção , RNA Ribossômico 16S/genética , Reprodutibilidade dos Testes
16.
Ticks Tick Borne Dis ; 10(6): 101261, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31337544

RESUMO

Rickettsia parkeri sensu stricto (s.s.) is an emerging human pathogen in the Americas. Comprehension of the etiology of R. parkeri infections in South America is complicated by the existence of genetic variants (Atlantic rainforest, NOD and Parvitarsum) of this species that are associated with specific groups of Amblyomma ticks. The rickettsial bacterium strain ApPR was first reported in Amblyomma parkeri ticks in Southern Brazil in 2012 and was considered, based on sequencing of fragments of the gltA, htrA, ompA and ompB genes, to represent yet another genetic variant of R. parkeri. In the current work, a multi-locus phylogenetic analysis employing additional genes and intragenic regions was performed using DNA extracted from (a) larvae of A. parkeri and Amblyomma species haplotype Nazaré ticks collected from wild birds, (b) a nymph of Amblyomma sp. haplotype Nazaré recovered from a monkey (Callicebus nigrifons), representing the first report of that tick parasitizing a non-human primate and (c) from a cultured isolate of ApPR, isolated from colony-reared adults of Amblyomma geayi. Phylogenetic inference performed using Maximum-likelihood (ML), Maximum Parsimony (MP) and Bayesian (B) methods, consistently placed strain ApPR outside the New World R. parkeri complex and instead grouped it in proximity to the Old World species Rickettsia africae and Rickettsia sibirica. Estimates of evolutionary divergence provided additional support for the inferred phylogenetic relationship. Given the clear evolutionary distance between strain ApPR and R. parkeri we propose the recognition of "Candidatus Rickettsia paranaensis".


Assuntos
Ixodidae/microbiologia , Rickettsia/classificação , Animais , Aves/microbiologia , Brasil , DNA Bacteriano/análise , DNA Intergênico/análise , Feminino , Ixodidae/crescimento & desenvolvimento , Larva/crescimento & desenvolvimento , Larva/microbiologia , Ninfa/crescimento & desenvolvimento , Ninfa/microbiologia , Filogenia , Pitheciidae/microbiologia , Rickettsia/genética , Análise de Sequência de DNA
17.
Ticks Tick Borne Dis ; 10(6): 101260, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31327747

RESUMO

In camels and their infesting ectoparasites, specific detection of pathogenic Anaplasma platys and genetically related strains (A. platys-like strains) remains problematic. This requires sequencing of the hemi-nested PCR products specific to A. platys and related strains. In this study, a PCR/RFLP method, earlier developed for specific detection of A. platys-like strains in animal species other than camels, was adapted in order to subtype A. platys-like strains isolated from camels and their ticks and to differentiate them from pathogenic A. platys without going through a sequencing step. This approach was used for investigating the infections with A. platys and related strains in 412 Camelus dromedarius camels and 334 feeding ticks from five Tunisian governorates. Microscopic examination using Giemsa-stained blood smears was performed in order to specify which types of cells were infected. Ticks were identified as Hyalomma dromedarii (n = 164, 49%), H. impeltatum (n = 161, 48.3%) and H. excavatum (n = 9, 2.7%). A. platys was not detected in any of the tested camels or ticks. The overall prevalence of A. platys-like strains was 5.6% (23/412) in camels and microscopic examination of infected cells showed a tropism for neutrophil granulocytes. One tick identified as H. dromedarii out of 327 analyzed ticks was found to be infected with A. platys-like strains (0.3%). Alignment, identity comparison and phylogenetic analysis of the 16S rRNA partial sequences obtained in this study suggest that Tunisian dromedaries and feeding ticks are infected with different Anaplasma strains genetically related to A. platys. Sequence analysis and phylogenetic study based on the groEL gene confirm the RFLP results and show that camel strains formed a separate sub-cluster relatively close to A. platys-like strains infecting Tunisian cattle. This adapted RFLP assay allows fast and specific detection of pathogenic A. platys and A. platys-like strains in camels and infesting ticks and has the intrinsic potential of revealing co-infections with these two types of bacteria in the same sample, reducing the time and costs associated with cloning and sequencing during molecular diagnosis.


Assuntos
Anaplasma/isolamento & purificação , Anaplasmose/epidemiologia , Camelus , Ixodidae/microbiologia , Infestações por Carrapato/veterinária , Anaplasma/genética , Anaplasmose/microbiologia , Animais , Proteínas de Bactérias/análise , Sequência de Bases , Chaperonina 60/análise , DNA Bacteriano/análise , Feminino , Ixodidae/fisiologia , Masculino , Filogenia , Prevalência , RNA Bacteriano/análise , RNA Ribossômico 16S/análise , Infestações por Carrapato/epidemiologia , Tunísia/epidemiologia
18.
Microbiol Immunol ; 63(10): 407-412, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31342547

RESUMO

Loop-mediated isothermal amplification (LAMP) assays are used to detect diverse pathogens. Initially, LAMP amplicons were detected using electrophoresis; later, real-time monitoring based on turbidity was developed to overcome the problem of contamination with environmental DNA. Recently, real-time monitoring of fluorescence signals using a quenching primer and probe has improved the reliability of amplification signals. Here, methods of detecting LAMP amplicons are reviewed.


Assuntos
Infecções Bacterianas/microbiologia , DNA Bacteriano/análise , DNA de Protozoário/análise , DNA Viral/análise , Técnicas de Amplificação de Ácido Nucleico/métodos , Infecções por Protozoários/parasitologia , Viroses/virologia , Fluorescência , Nefelometria e Turbidimetria/métodos , Reprodutibilidade dos Testes
19.
J Vet Med Sci ; 81(9): 1234-1237, 2019 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-31292334

RESUMO

Actinobacillus species are known to be pathogenic to horses. To clarify etiological agents of actinobacillosis in Japanese adult horses, 27 isolates from Japanese Thoroughbred racehorses putatively identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as Actinobacillus were further identified by PCR of the A. equuli toxin gene, by CAMP test, and by 16S rRNA sequencing analysis. Actinobacillus equuli subsp. haemolyticus was isolated most frequently (16/27) and was related to respiratory infections. Actinobacillus equuli subsp. equuli (4/27) was isolated from chronic cases or concomitant with other bacterial infections. The remainder were A. pleuropneumoniae, unclassified Actinobacillus species and Pasteurella caballi. Actinobacillus equuli including subsp. haemolyticus and subsp. equuli were the species most frequently isolated from equine actinobacillosis in Japan.


Assuntos
Infecções por Actinobacillus/veterinária , Actinobacillus/isolamento & purificação , Doenças dos Cavalos/microbiologia , Actinobacillus/classificação , Actinobacillus/genética , Animais , Proteínas de Bactérias/genética , DNA Bacteriano/análise , Doenças dos Cavalos/etiologia , Cavalos , Japão , Pasteurella/isolamento & purificação , Reação em Cadeia da Polimerase/veterinária , RNA Ribossômico 16S , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/veterinária
20.
Sci Total Environ ; 689: 789-796, 2019 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-31280161

RESUMO

The sesquiterpene geosmin, mainly originating from cyanobacteria, is considered one of the problematic odor compounds responsible for unpleasant-tasting and -smelling water episodes in freshwater supplies. The biochemistry and genetics of geosmin synthesis in cyanobacteria is well-elucidated and the geosmin synthase gene (geo) has been cloned and characterized in recent years. However, understanding the diversity, origin, and evolution of geo has been hindered by the limited availability of geo sequences to date. On the basis of the cloned geo sequences from16 filamentous geosmin-producing cyanobacterial species, representing 11 genera in Nostocales and Oscillatoriales, the diversity and evolution of geo in cyanobacteria was systematically analyzed in this study. Homologous alignment revealed that geo is highly conserved among the examined cyanobacterial species, with DNA sequence identities >0.72. Phylogenetic reconstruction and codon bias analysis based on geo suggest that cyanobacterial geo form a monophyletic branch with a common origin and ancestor for cyanobacteria, actinomycetes, and myxobacteria. The global ratio of nonsynonymous/synonymous nucleotide substitutions (dN/dS) was 0.125, which is substantially <1 and indicates strong purifying selection in the evolution of cyanobacterial geo. To add to further interest, horizontal gene transfer of cyanobacterial geo in evolutionary history was confirmed by the discovery of an incongruent coevolutionary relationship between geo and housekeeping genes 16S rDNA and rpoC. The present study enhances the fundamental understanding of cyanobacterial geo in diversity and evolution, and sheds light on the development of molecular assays for detection and molecular ecology research of geosmin-producing cyanobacteria.


Assuntos
Proteínas de Bactérias/genética , Cianobactérias/genética , Evolução Molecular , Variação Genética , Naftóis/metabolismo , Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , DNA Bacteriano/análise , Filogenia , Análise de Sequência de DNA , Especificidade da Espécie
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