Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 16.630
Filtrar
1.
Nat Commun ; 11(1): 4774, 2020 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-32963224

RESUMO

Detection of microbial nucleic acids in body fluids has become the preferred method for rapid diagnosis of many infectious diseases. However, culture-based diagnostics that are time-consuming remain the gold standard approach in certain cases, such as sepsis. New culture-free methods are urgently needed. Here, we describe Single MOLecule Tethering or SMOLT, an amplification-free and purification-free molecular assay that can detect microorganisms in body fluids with high sensitivity without the need of culturing. The signal of SMOLT is generated by the displacement of micron-size beads tethered by DNA probes that are between 1 and 7 microns long. The molecular extension of thousands of DNA probes is determined with sub-micron precision using a robust and rapid optical approach. We demonstrate that SMOLT can detect nucleic acids directly in blood, urine and sputum at sub-femtomolar concentrations, and microorganisms in blood at 1 CFU mL-1 (colony forming unit per milliliter) threefold faster, with higher multiplexing capacity and with a more straight-forward protocol than amplified methodologies. SMOLT's clinical utility is further demonstrated by developing a multiplex assay for simultaneous detection of sepsis-causing Candida species directly in whole blood.


Assuntos
Líquidos Corporais/química , Técnicas de Diagnóstico Molecular/métodos , Ácidos Nucleicos/isolamento & purificação , Sepse/diagnóstico , Candida/genética , Candida/isolamento & purificação , Candidíase/diagnóstico , Contagem de Colônia Microbiana , Doenças Transmissíveis/diagnóstico , DNA/isolamento & purificação , Humanos , Ácidos Nucleicos/sangue , Ácidos Nucleicos/urina , Reação em Cadeia da Polimerase/métodos , RNA/isolamento & purificação , Sensibilidade e Especificidade , Sepse/microbiologia , Infecções Estafilocócicas/diagnóstico , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Urina
2.
PLoS Negl Trop Dis ; 14(8): e0008609, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32822351

RESUMO

As a unique feature among otherwise hermaphroditic trematodes, Schistosoma species are gonochoric parasites whose sex is genetically determined (ZZ for males and ZW for females). However, schistosome larvae are morphologically identical, and sex can only be discriminated by molecular methods. Here, we integrated published Schistosoma. japonicum transcriptome and genome data to identify W chromosome-specific genes as sex biomarkers. Three W chromosome-specific genes of S. japonicum were identified as sex biomarkers from a panel of 12 genes expressed only in females. An efficient duplex real-time PCR (qPCR) method for sexing cercariae was developed which could identify the sex of cercariae within 2 h without DNA extraction. Moreover, this method can be used to identify not only single-sex but also mixed-sex schistosome-infected snails. We observed a nearly equal proportion of single-male, single-female, and mixed-sex schistosome infections in artificially infected snails. Sex-known schistosome-infected snail models can be efficiently constructed with the aid of duplex qPCR. A field study revealed that single-sex schistosome infections were predominant among naturally infected snails. Finally, a schistosomiasis mouse model based on sex-known cercariae infection was shown to be more reliable than a model based on sex-unknown cercariae infection. The developed duplex qPCR method for sexing S. japonicum cercariae can be widely used for schistosomiasis modeling, genetic experiments, and field-based molecular epidemiological studies.


Assuntos
Cercárias/genética , Cromossomos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Schistosoma japonicum/genética , Esquistossomose/parasitologia , Animais , Biomarcadores , China/epidemiologia , DNA/isolamento & purificação , Modelos Animais de Doenças , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Esquistossomose/epidemiologia , Caramujos/parasitologia
3.
PLoS Negl Trop Dis ; 14(8): e0008552, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32845881

RESUMO

BACKGROUND: Mitochondrial genomes provide useful genetic markers for systematic and population genetic studies of parasitic helminths. Although many such genome sequences have been published and deposited in public databases, there is evidence that some of them are incomplete relating to an inability of conventional techniques to reliably sequence non-coding (repetitive) regions. In the present study, we characterise the complete mitochondrial genome-including the long, non-coding region-of the carcinogenic Chinese liver fluke, Clonorchis sinensis, using long-read sequencing. METHODS: The mitochondrial genome was sequenced from total high molecular-weight genomic DNA isolated from a pool of 100 adult worms of C. sinensis using the MinION sequencing platform (Oxford Nanopore Technologies), and assembled and annotated using an informatic approach. RESULTS: From > 93,500 long-reads, we assembled a 18,304 bp-mitochondrial genome for C. sinensis. Within this genome we identified a novel non-coding region of 4,549 bp containing six tandem-repetitive units of 719-809 bp each. Given that genomic DNA from pooled worms was used for sequencing, some variability in length/sequence in this tandem-repetitive region was detectable, reflecting population variation. CONCLUSIONS: For C. sinensis, we report the complete mitochondrial genome, which includes a long (> 4.5 kb) tandem-repetitive region. The discovery of this non-coding region using a nanopore-sequencing/informatic approach now paves the way to investigating the nature and extent of length/sequence variation in this region within and among individual worms, both within and among C. sinensis populations, and to exploring whether this region has a functional role in the regulation of replication and transcription, akin to the mitochondrial control region in mammals. Although applied to C. sinensis, the technological approach established here should be broadly applicable to characterise complex tandem-repetitive or homo-polymeric regions in the mitochondrial genomes of a wide range of taxa.


Assuntos
Clonorchis sinensis/genética , Genoma Mitocondrial , Sequências de Repetição em Tandem/genética , Animais , Sequência de Bases , DNA/isolamento & purificação
4.
Sensors (Basel) ; 20(16)2020 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-32824787

RESUMO

Pandemics require a fast and immediate response to contain potential infectious carriers. In the recent 2020 Covid-19 worldwide pandemic, authorities all around the world have failed to identify potential carriers and contain it on time. Hence, a rapid and very sensitive testing method is required. Current diagnostic tools, reverse transcription PCR (RT-PCR) and real-time PCR (qPCR), have its pitfalls for quick pandemic containment such as the requirement for specialized professionals and instrumentation. Versatile electrochemical DNA/RNA sensors are a promising technological alternative for PCR based diagnosis. In an electrochemical DNA sensor, a nucleic acid hybridization event is converted into a quantifiable electrochemical signal. A critical challenge of electrochemical DNA sensors is sensitive detection of a low copy number of DNA/RNA in samples such as is the case for early onset of a disease. Signal amplification approaches are an important tool to overcome this sensitivity issue. In this review, the authors discuss the most recent signal amplification strategies employed in the electrochemical DNA/RNA diagnosis of pathogens.


Assuntos
Betacoronavirus/isolamento & purificação , Técnicas Biossensoriais , Infecções por Coronavirus/diagnóstico , Técnicas Eletroquímicas , Pneumonia Viral/diagnóstico , Betacoronavirus/patogenicidade , Infecções por Coronavirus/epidemiologia , Infecções por Coronavirus/virologia , DNA/isolamento & purificação , Epidemias/prevenção & controle , Humanos , Pandemias , Pneumonia Viral/epidemiologia , Pneumonia Viral/virologia , RNA/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real
5.
PLoS One ; 15(7): e0235222, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32639972

RESUMO

Here we present and justify an approach for minimal-destructive DNA extraction from historic insect specimens for next generation sequencing applications. An increasing number of studies use insects from museum collections for biodiversity research. However, the availability of specimens for molecular analyses has been limited by the degraded nature of the DNA gained from century-old museum material and the consumptive nature of most DNA extraction procedures. The method described in this manuscript enabled us to successfully extract DNA from specimens as old as 241 years using a minimal-destructive approach. The direct comparison of the DNeasy extraction Kit and the Monarch® PCR & DNA Clean-up Kit showed a significant increase of 17.3-fold higher DNA yield extracted with the Monarch Oligo protocol on average. By using an extraction protocol originally designed for oligonucleotide clean-up, we were able to combine overcoming the restrictions by target fragment size and strand state, with minimising time consumption and labour-intensity. The type specimens used for the minimal-destructive DNA extraction exhibited no significant external change or post-extraction damage, while sufficient DNA was retrieved for analyses.


Assuntos
DNA/isolamento & purificação , Insetos/genética , Animais , DNA/genética , Fósseis/anatomia & histologia , Sequenciamento de Nucleotídeos em Larga Escala , Insetos/anatomia & histologia , Insetos/química , Filogenia
6.
Nucleic Acids Res ; 48(15): e86, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32544240

RESUMO

Specific nucleic acid sequences can be detected in individual cells by in situ hybridization. However, when very few copies of a target sequence are present per cell, its signal is undetectable by flow cytometry. Although various approaches have been developed to increase fluorescence signals for in situ hybridization, flow cytometric detection of specific genomic DNA sequences has not been established. Here, we present a flow cytometry assay for detection of single-copy genomic sequences in human lymphocytes using in situ PCR with universal energy transfer-labelled primers.


Assuntos
DNA/isolamento & purificação , Citometria de Fluxo/métodos , Imagem Individual de Molécula/métodos , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Humanos , Hibridização In Situ/métodos , Linfócitos/química , Reação em Cadeia da Polimerase/métodos
7.
Nucleic Acids Res ; 48(15): e89, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32544247

RESUMO

Understanding the thermodynamics of DNA motifs is important for prediction and design of probes and primers, but melt curve analyses are low-throughput and produce inaccurate results for motifs such as bulges and mismatches. Here, we developed a new, accurate and high-throughput method for measuring DNA motif thermodynamics called TEEM (Toehold Exchange Energy Measurement). It is a refined framework of comparing two toehold exchange reactions, which are competitive strand displacement between oligonucleotides. In a single experiment, TEEM can measure over 1000 ΔG° values with standard error of roughly 0.05 kcal/mol.


Assuntos
DNA/isolamento & purificação , Ensaios de Triagem em Larga Escala/métodos , Oligonucleotídeos/genética , Termodinâmica , DNA/química , Humanos , Conformação de Ácido Nucleico , Motivos de Nucleotídeos/genética , Oligonucleotídeos/química
8.
PLoS One ; 15(5): e0233573, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32437469

RESUMO

The accuracy of the DNA barcoding tool depends on the existence of a comprehensive archived library of sequences reliably determined at species level by expert taxonomists. However, misidentifications are not infrequent, especially following large-scale DNA barcoding campaigns on diverse and taxonomically complex groups. In this study we used the species-rich flea beetle genus Longitarsus, that requires a high level of expertise for morphological species identification, as a case study to assess the accuracy of the DNA barcoding tool following several optimization procedures. We built a cox1 reference database of 1502 sequences representing 78 Longitarsus species, among which 117 sequences (32 species) were newly generated using a non-invasive DNA extraction method that allows keeping reference voucher specimens. Within this dataset we identified 69 taxonomic inconsistencies using barcoding gap analysis and tree topology methods. Threshold optimisation and a posteriori taxonomic revision based on newly generated reference sequences and metadata allowed resolving 44 sequences with ambiguous and incorrect identification and provided a significant improvement of the DNA barcoding accuracy and identification efficacy. Unresolved taxonomic uncertainties, due to overlapping intra- and inter-specific levels of divergences, mainly regards the Longitarsus pratensis species complex and polyphyletic groups L. melanocephalus, L. nigrofasciatus and L. erro. Such type of errors indicates either poorly established taxonomy or any biological processes that make mtDNA groups poorly predictive of species boundaries (e.g. recent speciation or interspecific hybridisation), thus providing directions for further integrative taxonomic and evolutionary studies. Overall, this study underlines the importance of reference vouchers and high-quality metadata associated to sequences in reference databases and corroborates, once again, the key role of taxonomists in any step of the DNA barcoding pipeline in order to generate and maintain a correct and functional reference library.


Assuntos
Besouros/genética , Código de Barras de DNA Taxonômico , Animais , Besouros/classificação , DNA/genética , DNA/isolamento & purificação , Código de Barras de DNA Taxonômico/métodos , Bases de Dados de Ácidos Nucleicos , Evolução Molecular
9.
Nat Commun ; 11(1): 2590, 2020 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-32444602

RESUMO

A fundamental goal in microbiome studies is determining which microbes affect host physiology. Standard methods for determining changes in microbial taxa measure relative, rather than absolute abundances. Moreover, studies often analyze only stool, despite microbial diversity differing substantially among gastrointestinal (GI) locations. Here, we develop a quantitative framework to measure absolute abundances of individual bacterial taxa by combining the precision of digital PCR with the high-throughput nature of 16S rRNA gene amplicon sequencing. In a murine ketogenic-diet study, we compare microbial loads in lumenal and mucosal samples along the GI tract. Quantitative measurements of absolute (but not relative) abundances reveal decreases in total microbial loads on the ketogenic diet and enable us to determine the differential effects of diet on each taxon in stool and small-intestine mucosa samples. This rigorous quantitative microbial analysis framework, appropriate for diverse GI locations enables mapping microbial biogeography of the mammalian GI tract and more accurate analyses of changes in microbial taxa in microbiome studies.


Assuntos
Dieta Cetogênica , Microbioma Gastrointestinal/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mucosa Intestinal/microbiologia , Reação em Cadeia da Polimerase/métodos , Animais , DNA/isolamento & purificação , Fezes/microbiologia , Feminino , Dispositivos Lab-On-A-Chip , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase/instrumentação , RNA Ribossômico 16S , Verrucomicrobia/genética , Fluxo de Trabalho
10.
Toxicol Lett ; 331: 75-81, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32434050

RESUMO

Fungi of the genus Alternaria infest many agricultural crops and produce numerous mycotoxins, of which altertoxin II (ATX II) is one of the most mutagenic metabolites. ATX II carries an epoxide group but the formation of DNA adducts has not been demonstrated to date. We report now that ATX II gives rise to two covalent adducts with guanine when incubated with DNA under cell-free conditions. These adducts were demonstrated by LC-high resolution MS after enzymatic degradation of the incubated DNA to deoxynucleosides. The major adduct results from the covalent binding of ATX II, presumably through the epoxide group, to guanine, whereas the minor guanine adduct is derived from the major one by the elimination of two equivalents of water. In addition, a third adduct was detected, formed through covalent binding of ATX II to cytosine followed by the loss of two equivalents of water. The direct DNA reactivity of ATX II may explain its high mutagenicity.


Assuntos
Benzo(a)Antracenos/toxicidade , Adutos de DNA/análise , DNA/química , Guanina/química , Mutagênicos/toxicidade , Alternaria/química , Animais , Benzo(a)Antracenos/isolamento & purificação , Cromatografia Líquida , DNA/isolamento & purificação , Masculino , Espectrometria de Massas , Salmão , Testículo
11.
J Parasitol ; 106(2): 261-267, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32294757

RESUMO

The genus Rhabdochona Railliet, 1916 is one of the most speciose group of nematodes, parasites of freshwater fishes, with approximately 103 species described worldwide. Twenty-two species have been recorded in the Americas, 14 of them in Mexico. In this paper we describe a new species of Rhabdochona on the basis of light and scanning electron microscopy and molecular tools. Rhabdochona adentata n. sp. was recovered from the gallbladder of the freshwater Oaxaca killifish, Profundulus oaxacae (Meek, 1902) (Profundulidae) captured in the Río Grande, State of Oaxaca, Mexico. Rhabdochona adentata n. sp. differs markedly from its congeners by possessing an unusual prostom lacking anterior teeth, small simple deirids, and the location of the excretory pore at the level of the union of the muscular and glandular esophagus. Sequences of cytochrome c oxidase subunit I (COI) from mitochondrial deoxyribonucleic acid and the D2 + D3 domains of the large ribosomal subunit (28S) were obtained from 3 specimens and were analyzed using maximum likelihood and Bayesian inference analyses. Phylogenetic analyses using COI and 28S genes recovered 1 new lineage of Rhabdochona. The new species is described on the basis of a detailed morphological study. This parasite represents the first species of Rhabdochona without prostomal teeth and with a different site of infection, the gallbladder.


Assuntos
Ciprinodontiformes/parasitologia , Doenças dos Peixes/parasitologia , Infecções por Spirurida/veterinária , Spiruroidea/classificação , Animais , Teorema de Bayes , DNA/química , DNA/isolamento & purificação , Complexo IV da Cadeia de Transporte de Elétrons/genética , Feminino , Doenças dos Peixes/epidemiologia , Água Doce , Vesícula Biliar/parasitologia , Funções Verossimilhança , Masculino , México/epidemiologia , Microscopia Eletrônica de Varredura/veterinária , Filogenia , Prevalência , RNA Ribossômico 28S/genética , Rios , Infecções por Spirurida/epidemiologia , Infecções por Spirurida/parasitologia , Spiruroidea/genética , Spiruroidea/ultraestrutura
12.
Anthropol Anz ; 77(3): 235-242, 2020 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-32211746

RESUMO

This paper discusses our approach and results obtained when attempting to identify a saponified human body recovered from the sea, without arms and legs. Bones, especially the long ones, are the only sources of DNA available in several cases involving unidentified bodies in advanced state of putrefaction. In this case, since the body was found without limbs, attempts were made to extract DNA from the sternum bone. The DNA was extracted using a modified version of the NucleoSpin® DNA Trace Kit (Macherey Nagel™) protocol and an STR analysis was performed. Thanks to this modified protocol a complete DNA profile was obtained from the sternum bone, while only partial results were obtained from blood and teeth. The DNA profile obtained from the sternum was compared with the DNA of the putative son searching for a genetic match. Five incompatibilities were detected so it was possible to exclude the kinship. In conclusion this could be a useful technique in personal identification through DNA analysis in case of poor quality and quantity of bone.


Assuntos
Impressões Digitais de DNA , DNA , Dente , Antropologia Física , DNA/isolamento & purificação , Humanos , Repetições de Microssatélites , Esterno
13.
PLoS One ; 15(3): e0229353, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32163447

RESUMO

In the last few years, DNA barcoding became an established method for species identification in biodiversity inventories and monitoring studies. Such studies depend on the access to a comprehensive reference data base, covering all relevant taxa. Here we present a comprehensive DNA barcode inventory of all amphibian and reptile species native to Austria, except for the putatively extinct Vipera ursinii rakosiensis and Lissotriton helveticus, which has been only recently reported for the very western edge of Austria. A total of 194 DNA barcodes were generated in the framework of the Austrian Barcode of Life (ABOL) initiative. Species identification via DNA barcodes was successful for most species, except for the hybridogenetic species complex of water frogs (Pelophylax spp.) and the crested newts (Triturus spp.), in areas of sympatry. However, DNA barcoding also proved powerful in detecting deep conspecific lineages, e.g. within Natrix natrix or the wall lizard (Podarcis muralis), resulting in more than one Barcode Index Number (BIN) per species. Moreover, DNA barcodes revealed the presence of Natrix helvetica, which has been elevated to species level only recently, and genetic signatures of the Italian water frog Pelophylax bergeri in Western Austria for the first time. Comparison to previously published DNA barcoding data of European amphibians and reptiles corroborated the results of the Austrian data but also revealed certain peculiarities, underlining the particular strengths and in the case of the genus Pelophylax also the limitations of DNA barcoding. Consequently, DNA barcoding is not only powerful for species identification of all life stages of most Austrian amphibian and reptile species, but also for the detection of new species, the monitoring of gene flow or the presence of alien populations and/or species. Thus, DNA barcoding and the data generated in this study may serve both scientific and national or even transnational conservation purposes.


Assuntos
Anfíbios/genética , Biodiversidade , Código de Barras de DNA Taxonômico/métodos , DNA/genética , Biblioteca Gênica , Répteis/genética , Animais , Áustria , DNA/isolamento & purificação , Filogenia , Padrões de Referência , Especificidade da Espécie
14.
J Parasitol ; 106(2): 221-232, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-32164028

RESUMO

Members of the sucking louse genus Pedicinus are ectoparasites of cercopithecid primates in Africa, Asia, and Gibraltar. Pedicinus gabonensis n. sp. is described on the basis of adult male and female specimens collected from the mandrill (Mandrillus sphinx) in Gabon. The new species is compared morphologically with other members of the genus Pedicinus, and a nuclear elongation factor 1 alpha gene sequence is provided. Host associations and geographical distributions of the 18 previously recognized species of the genus and of P. gabonensis n. sp. are reviewed. Updated identification keys are provided for males and females of all known valid species of Pedicinus.


Assuntos
Anoplura/classificação , Infestações por Piolhos/veterinária , Mandrillus/parasitologia , Doenças dos Macacos/parasitologia , Animais , Anoplura/anatomia & histologia , Anoplura/genética , Anoplura/fisiologia , DNA/química , DNA/isolamento & purificação , Feminino , Gabão/epidemiologia , Infestações por Piolhos/epidemiologia , Infestações por Piolhos/parasitologia , Masculino , Doenças dos Macacos/epidemiologia , Alinhamento de Sequência/veterinária , Análise de Sequência de DNA/veterinária
15.
Mol Biol Rep ; 47(3): 2405-2413, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32020430

RESUMO

Bacterial artificial chromosome (BAC) library is an important genomic resource useful in targeted marker development, positional cloning, physical mapping and a substrate for genome sequencing for better understanding the genome organization of a species. The present manuscript elucidates the improvement in protocols for economical and efficient BAC insert DNA isolation, BAC end sequencing and FISH for physical localization on the metaphase chromosome complements. BAC clones of Clarias magur, maintained in 384-well plate format in our laboratory, were used in this study. The protocols gave consistent and efficient results. We use routinely these protocols for BAC insert DNA extraction, generating end sequence data of the clone and constructing DNA probes to hybridize on the metaphase spreads of C. magur using FISH for physical their localization.


Assuntos
Cromossomos Artificiais Bacterianos , DNA/isolamento & purificação , Hibridização in Situ Fluorescente , Mapeamento Físico do Cromossomo , Análise de Sequência de DNA , Biologia Computacional/métodos , Biblioteca Gênica , Hibridização in Situ Fluorescente/métodos , Mapeamento Físico do Cromossomo/métodos , Análise de Sequência de DNA/métodos , Software
16.
Parasitol Res ; 119(3): 1109-1115, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32086592

RESUMO

Black bears (Ursus americanus) are commonly exposed to Toxoplasma gondii. However, there are no reports of exposure or infection with T. gondii in black bears from Oklahoma. The purpose of our project was to determine the seroprevalence of T. gondii antibodies in black bears collected in Oklahoma. Additionally, since only serum was available from these bears, we sought to determine if DNA extraction and PCR amplification for T. gondii was possible on serum samples from bears with positive titers. Seroprevalence was determined using modified agglutination test (MAT). Serum was collected from 44 live-trapped bears in southeastern Oklahoma; 32 (73% ± 58-84%) had antibodies against T. gondii. Seroprevalence in adult bears (85% ± 67-95%) was significantly higher (p = 0.028) than yearlings (33.0% ± 56-80%). Adult bears were 3.4 times more likely to have antibodies to T. gondii than yearlings. From the bears with positive titers, T. gondii DNA was detected in 12 of the 32 seropositive samples by PCR of the B1 gene, with two of the samples showing variation in two nucleotide positions when compared with available sequences. Multilocus PCR-RFLP genotyping of these 12 samples revealed three ToxoDB genotypes, including #2 (type III, haplogroup 3), #4 (type XII, haplogroup 12), and #74 (haplogroup 12). To the best of our knowledge, this is the first report of T. gondii seroprevalence in black bears from Oklahoma. Our results indicate that exposure and infection with T. gondii in black bears from Oklahoma is common.


Assuntos
Anticorpos Antiprotozoários/sangue , Toxoplasma/genética , Toxoplasmose Animal/epidemiologia , Toxoplasmose Animal/parasitologia , Ursidae/parasitologia , Testes de Aglutinação , Animais , DNA/isolamento & purificação , Genótipo , Oklahoma/epidemiologia , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Estudos Soroepidemiológicos
17.
J Parasitol ; 106(1): 14-24, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31958374

RESUMO

Human head lice and body lice (Pediculus humanus) are neglected ectoparasites. Head lice continue to be prevalent in children worldwide, and insecticide resistance in these insects has complicated their treatment. Meanwhile, body lice, which are most common in the developing world, are resurging among marginalized populations in developed nations. Today, the microbiome is being increasingly recognized as a key mediator of insect physiology. However, the microbial communities that inhabit human lice have remained unknown beyond only a few species of bacteria. Knowledge of the microbiomes of head and body lice could improve our understanding of the observed physiological differences between the 2 ecotypes and potentially inform the development of novel interventions against lice infestations and louse-borne infectious diseases. Toward these goals, here we performed 16S rRNA gene amplicon sequencing to characterize the microbiomes of both head and body lice and identify patterns of interest among these communities. Our data reveal that head and body lice harbor limited but distinct communities of bacteria that include known intracellular endosymbionts ("Candidatus Riesia pediculicola"), extracellular bacteria that may be horizontally acquired from the host environment, and a number of taxa of known or potential public health significance. Notably, in body lice, the relative abundance of vertically transmitted endosymbionts is lower than in head lice, which is a significant driver of greater alpha diversity. Further, several differentially abundant non-endosymbiont taxa and differences in beta diversity were observed between head lice and body lice. These findings support the hypothesis that microbiome differences could contribute to the divergence between human louse ecotypes and underscore the need for future studies to better comprehend the acquisition and physiological roles of human lice microbiomes.


Assuntos
Bactérias/classificação , Ecótipo , Microbiota , Pediculus/microbiologia , RNA Ribossômico 16S/química , Animais , Bactérias/genética , DNA/isolamento & purificação , Feminino , Humanos , Pediculus/classificação , Pediculus/fisiologia , Análise de Componente Principal , Coelhos , Análise de Sequência de RNA
18.
Malar J ; 19(1): 29, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31952536

RESUMO

BACKGROUND: Anopheles maculipennis complex, the historic vector of malaria, causes serious medical problems worldwide and exhibits different behaviours. Studying the odorant-binding proteins (OBPs), which influence the chemosensory system and behavioural responses, is essential to understanding the population structure and developing effective control measures against this vector. The present study was designed to identify and analyse the obp1 gene in An. maculipennis. METHODS: Adults of An. maculipennis sensu stricto were collected in Zanjan Province, northwest of Iran, and gDNAs of female mosquitoes were extracted. Fragments of An. maculipennis obp1 (Amacobp1) gene were amplified using degenerate and specific primers, and some of amplicons were selected for sequencing. RESULTS: Analysis of amplified products identified that the sequence of Amacobp1 gene was 1341 bp long. This gene contains three exons (5', internal, and 3'of 160, 256, and 18 bp, respectively) and encodes 144 amino acids. The sizes of introns I and II in deduced gene are 268 and 358 nucleotides, respectively. The amino acid sequence in the C-terminal of AmacOBP1 is similar to that of major malaria vector Anopheles species. However, its N-terminal has a specific signal peptide with 19 amino acids. This peptide is conserved in different studied populations, and its sequence of amino acids shows the most variation among anopheline species. CONCLUSIONS: Degenerate primers in this study are suggested for studying obp1 gene in Anopheles species. Amacobp1 gene is proposed as a molecular marker for the detection of intraspecific ecotypes and diagnosis of different species within Maculipennis Group. Moreover, the N-terminal of AmacOBP1 peptide is recommended as a molecular marker to identify the Amacobp1 expression patterns in different chemosensory organs for assessing the molecular mechanisms and developing novel behavioural disturbance agents to control An. maculipennis.


Assuntos
Anopheles/química , Mosquitos Vetores/química , Receptores Odorantes/genética , Sequência de Aminoácidos , Animais , Anopheles/classificação , Anopheles/genética , Sequência de Bases , DNA/química , DNA/genética , DNA/isolamento & purificação , Éxons , Feminino , Íntrons , Irã (Geográfico) , Masculino , Mosquitos Vetores/classificação , Mosquitos Vetores/genética , Filogenia , Sinais Direcionadores de Proteínas/genética , Sinais Direcionadores de Proteínas/fisiologia , Receptores Odorantes/química , Alinhamento de Sequência
19.
J Clin Pathol ; 73(8): 514-518, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31919142

RESUMO

Genomic technologies are increasingly used clinically for both diagnosis and guiding cancer therapy. However, formalin fixation can compromise DNA quality. This study aimed to optimise tissue fixation using normal colon, liver and uterus (n=8 each) by varying neutral buffered formalin (NBF) concentration (1%-5% w/v) and fixation time (24-48 hours). Fixation using 4% NBF improved DNA quality (assessed by qPCR) compared with routine (4% unbuffered formal saline-fixed) specimens (p<0.01). Further improvements were achieved by reducing NBF concentration (p<0.00001), whereas fixation time had no effect (p=0.110). No adverse effects were detected by histopathological or QuPath morphometric analysis. Immunohistochemistry for multicytokeratin and α-smooth muscle actin revealed no changes in staining specificity or intensity in any tissue other than on liver multicytokeratin staining intensity, where the effect of fixation time was more significant (p=0.0004) than NBF concentration (p=0.048). Thus, reducing NBF concentration can maximise DNA quality without compromising tissue morphology or standard histopathological analyses.


Assuntos
DNA/isolamento & purificação , Fixadores/farmacologia , Formaldeído/farmacologia , Inclusão em Parafina/normas , Doenças do Colo/patologia , Feminino , Humanos , Imuno-Histoquímica/normas , Hepatopatias/patologia , Melhoria de Qualidade , Coloração e Rotulagem/normas , Fixação de Tecidos/normas , Doenças Uterinas/patologia
20.
Parasit Vectors ; 13(1): 33, 2020 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-31959216

RESUMO

BACKGROUND: Triatomines are natural vectors of Chagas disease and are mainly prevalent in the Americas. In China, previous data from decades ago showed that there were two species of triatomine bugs, Triatoma rubrofasciata and T. sinica. However, the distribution, genetic characteristics and public health implications of triatomines in China are still relatively unknown. In order to gain knowledge on the distribution, genetic characteristics and public health implications of the triatomines in Guangxi, China, an entomological-epidemiological study and genetic research was conducted. METHODS: Different methods were used to elucidate the distribution of triatomines in Guangxi including consultations with county-level Center for Disease Prevention and Control staff and village doctors, the distribution of educational material on triatomines though the internet and social media apps such as Wechat and QQ, and conducting manual inspections and light trapping to collect triatomines. The morphological characteristics of the collected triatomines were identified under light microscopy. The mitochondrial 16S rRNA, cytochrome b (cytb) genes and nuclear 28S rRNA gene were amplified, sequenced and used in phylogenetic analyses. RESULTS: A total of 305 triatomines were captured from 54 different sites in 13 cities in Guangxi. All collected bugs were identified as T. rubrofasciata based on morphology. Most triatomine collection sites were around or inside houses. Four triatomines bite cases were observed during the investigation indicating that triatomine bites are common, the bites can cause serious anaphylaxis and skin papules and urticaria, suggesting a systemic skin response. The 16S rRNA, 28S rRNA and cytb sequence analyses of T. rubrofasciata from Guangxi and other countries showed that T. rubrofasciata sequences from different regions exhibit a high similarity, with no geographical differences. The phylogenetic tree based on the 16S rRNA and cytb genes showed that T. rubrofasciata sequences from different regions and continents were in the same cluster, indicating no differentiation among different geographical populations. CONCLUSIONS: Our study showed that T. rubrofasciata is widely distributed in Guangxi and that people are commonly bitten by this insect in some regions. This highlights the need to enhance surveillance for and control of T. rubrofasciata and to strengthen the monitoring of imported Trypanosoma cruzi in China. The 16S rRNA, 28S rRNA and cytb sequence analyses of T. rubrofasciata from different regions and continents suggested that T. rubrofasciata populations exhibit high similarity, and the clustering in the phylogenetic analyses indicates that T. rubrofasciata has a close ancestor originating in the Americas.


Assuntos
Doença de Chagas/transmissão , Mordeduras e Picadas de Insetos/epidemiologia , Insetos Vetores/fisiologia , Triatoma/fisiologia , Adulto , Animais , Doença de Chagas/epidemiologia , China/epidemiologia , Citocromos b/genética , DNA/química , DNA/isolamento & purificação , DNA Ribossômico/química , DNA Ribossômico/isolamento & purificação , Feminino , Habitação , Humanos , Mordeduras e Picadas de Insetos/parasitologia , Mordeduras e Picadas de Insetos/patologia , Insetos Vetores/classificação , Insetos Vetores/genética , Insetos Vetores/parasitologia , Masculino , Pessoa de Meia-Idade , Filogenia , RNA Ribossômico 16S/genética , RNA Ribossômico 28S/genética , Alinhamento de Sequência , Triatoma/classificação , Triatoma/genética , Triatoma/parasitologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA