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1.
Anal Chim Acta ; 1316: 342838, 2024 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-38969428

RESUMO

The diagnosis of dengue virus (DENV) has been challenging particularly in areas far from clinical laboratories. Early diagnosis of pathogens is a prerequisite for the timely treatment and pathogen control. An ideal diagnostic for viral infections should possess high sensitivity, specificity, and flexibility. In this study, we implemented dual amplification involving Cas13a and Cas12a, enabling sensitive and visually aided diagnostics for the dengue virus. Cas13a recognized the target RNA by crRNA and formed the assembly of the Cas13a/crRNA/RNA ternary complex, engaged in collateral cleavage of nearby crRNA of Cas12a. The Cas12a/crRNA/dsDNA activator ternary complex could not be assembled due to the absence of crRNA of Cas12a. Moreover, the probe, with 5' and 3' termini labeled with FAM and biotin, could not be separated. The probes labeled with FAM and biotin, combined the Anti-FAM and the Anti-Biotin Ab-coated gold nanoparticle, and conformed sandwich structure on the T-line. The red line on the paper strip caused by clumping of AuNPs on the T-line indicated the detection of dengue virus. This technique, utilizing an activated Cas13a system cleaving the crRNA of Cas12a, triggered a cascade that amplifies the virus signal, achieving a low detection limit of 190 fM with fluorescence. Moreover, even at 1 pM, the red color on the T-line was easily visible by naked eyes. The developed strategy, incorporating cascade enzymatic amplification, exhibited good sensitivity and may serve as a field-deployable diagnostic tool for dengue virus.


Assuntos
Vírus da Dengue , Vírus da Dengue/isolamento & purificação , Dengue/diagnóstico , Humanos , Técnicas de Amplificação de Ácido Nucleico/métodos , RNA Viral/análise , Proteínas Associadas a CRISPR/metabolismo , Nanopartículas Metálicas/química , Limite de Detecção , Ouro/química , Proteínas de Bactérias , Endodesoxirribonucleases
2.
Vet Res Forum ; 15(4): 171-179, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38770198

RESUMO

Rhipicephalus sanguineus, a repulsive obligate blood feeder, is a three-host tick inflicting tremendous damage. Blood-sucking initiates tick-pathogen-host interactions along with alterations in the expression levels of numerous bioactive ingredients. Key molecules regulating blood meals were identified using the transcriptomic approach. A total number of 744 transcripts showed statistically significantly differential expression including 309 significantly upregulated transcripts and 435 significantly downregulated transcripts in semiengorged female ticks compared to unfed ticks, all collected in 2021. The top 10 differentially upregulated transcripts with explicit functional annotations included turripeptide OL55-like protein, valine tRNA ligase-like protein and ice-structuring glycoprotein-like protein. The top 10 differentially down-regulated transcripts were uncharacterized proteins. Gene Ontology (GO) enrichment analysis revealed four associated terms in the cellular component category and 16 in the molecular function category among the top 20 terms. Differentially expressed genes (DEGs) were enriched in GO terms ID 0000323 (lytic vacuole) and ID 0005773 (vacuole). The top 20 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways included metabolism, cellular processes, organismal systems and human diseases. The DEGs were enriched in the KEGG term ID: ko-04142 (lysosome pathway) associated with intracellular digestion in the tick midgut epithelium. Molecular markers annotated via comparative transcriptomic profiling were expected to be candidate markers for the purpose of tick control.

3.
Emerg Infect Dis ; 30(6): 1249-1252, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38782141

RESUMO

Burkholderia semiarida was previously identified solely as a plant pathogen within the Burkholderia cepacia complex. We present a case in China involving recurrent pneumonia attributed to B. semiarida infection. Of note, the infection manifested in an immunocompetent patient with no associated primary diseases and endured for >3 years.


Assuntos
Infecções por Burkholderia , Burkholderia , Recidiva , Humanos , Infecções por Burkholderia/diagnóstico , Infecções por Burkholderia/microbiologia , Infecções por Burkholderia/tratamento farmacológico , China , Burkholderia/isolamento & purificação , Burkholderia/genética , Masculino , Imunocompetência , Antibacterianos/uso terapêutico , Pessoa de Meia-Idade , Pneumonia Bacteriana/microbiologia , Pneumonia Bacteriana/diagnóstico , Pneumonia Bacteriana/tratamento farmacológico
4.
Biosens Bioelectron ; 257: 116334, 2024 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-38678788

RESUMO

Burkholderia pseudomallei, widely distributed in tropical and subtropical ecosystems, is capable of causing the fatal zoonotic disease melioidosis and exhibiting a global trend of dissemination. Rapid and sensitive detection of B. pseudomallei is essential for environmental monitoring as well as infection control. Here, we developed an innovative biosensor for quantitatively detecting B. pseudomallei relies on ATP released triggered by bacteriophage-induced bacteria lysis. The lytic bacteriophage vB_BpP_HN01, with high specificity, is employed alongside magnetic nanoparticles assembly to create a biological receptor, facilitating the capture and enrichment of viable target bacteria. Following a brief extraction and incubation process, the captured target undergoes rapid lysis to release contents including ATP. The EXPAR-CRISPR cascade reaction provides an efficient signal transduction and dual amplification module that allowing the generated ATP to guide the signal output as an activator, ultimately converting the target bacterial amount into a detectable fluorescence signal. The proposed bacteriophage affinity strategy exhibited superior performance for B. pseudomallei detection with a dynamic range from 10^2 to 10^7 CFU mL-1, and a LOD of 45 CFU mL-1 within 80 min. Moreover, with the output signal compatible across various monitoring methods, this work offers a robust assurance for rapid diagnosis and on-site environmental monitoring of B. pseudomallei.


Assuntos
Trifosfato de Adenosina , Bacteriófagos , Técnicas Biossensoriais , Burkholderia pseudomallei , Sistemas CRISPR-Cas , Burkholderia pseudomallei/virologia , Técnicas Biossensoriais/métodos , Bacteriófagos/química , Bacteriófagos/isolamento & purificação , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/análise , Melioidose/microbiologia , Limite de Detecção , Humanos , Nanopartículas de Magnetita/química
5.
Parasit Vectors ; 17(1): 107, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38444018

RESUMO

BACKGROUND: Ticks serve as vectors for a diverse array of pathogens, including viruses responsible for both human and livestock diseases. Symbiotic bacteria hold significant potential for controlling tick-borne disease. However, the alteration of tick gut bacterial community in response to pathogen infection has not been analyzed for any tick-borne viruses. Here, the impact of severe fever with thrombocytopenia syndrome virus (SFTSV) infection on bacterial diversity in the gut of Haemaphysalis longicornis is investigated. METHODS: Unfed tick females were artificially infected with SFTSV. The gut samples were collected and the genomic DNA was extracted. We then investigated alterations in gut bacterial composition in response to SFTSV infection through 16S rRNA gene sequencing. RESULTS: The study found that a reduction in the number of operational taxonomic units (OTUs) in the tick gut following SFTSV infection. However, there were no significant changes in alpha diversity indices upon infection. Four genera, including Corynebacterium, Arthrobacter, Sphingomonas, and Escherichia, were identified as biomarkers for the tick gut without SFTSV infection. Notably, the predicted correlation network indicated that the biomarkers Sphingomonas and Escherichia exhibited positive correlations within the same subcommunity, which was altered upon viral infection. CONCLUSIONS: These findings revealed that the change in tick gut bacterial composition upon SFTSV infection and could facilitate the discovery new target for tick-borne viral disease control.


Assuntos
Microbioma Gastrointestinal , Febre Grave com Síndrome de Trombocitopenia , Feminino , Humanos , Animais , Haemaphysalis longicornis , RNA Ribossômico 16S/genética , Biomarcadores
6.
J Phys Condens Matter ; 36(17)2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38237180

RESUMO

High-quality epitaxial graphene (EG) on SiC is crucial to high-performance electronic devices due to the good compatibility with Si-based semiconductor technology. Metal intercalation has been considered as a basic technology to modify EG on SiC. In the past ten years, there have been extensive research activities on the structural evolution during EG fabrication, characterization of the atomic structure and electronic states of EG, optimization of the fabrication process, as well as modification of EG by metal intercalation. In this perspective, the developments and breakthroughs in recent years are summarized and future expectations are discussed. A good understanding of the growth mechanism of EG and subsequent metal intercalation effects is fundamentally important.

7.
Microbiol Spectr ; 11(6): e0112523, 2023 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-37815337

RESUMO

IMPORTANCE: This study focused on the development of a reaction system using rhPCR to amplify a specific gene, ORF2, of B. pseudomallei and to identify the P174L mutation associated with increased drug resistance to ceftazidime (CAZ). The system incorporated universal primer probes and a simple temperature cycle reaction. The amplified products were then analyzed using lateral flow strip assay (LFSA) for strain identification and mutation interpretation. The developed system provides a reliable basis for diagnosing melioidosis and selecting appropriate drugs. Its potential impact is particularly significant in resource-limited settings where access to advanced diagnostic techniques is limited. This platform stands out for its simplicity, convenience, sensitivity, specificity, and portability. It shows promise as a point-of-care testing method for detecting single nucleotide polymorphism in genes associated with other diseases. By leveraging the advantages of this platform, researchers and healthcare professionals can potentially expand its use beyond melioidosis and apply it to the rapid detection of genetic variations in other disease-related genes.


Assuntos
Burkholderia pseudomallei , Melioidose , Humanos , Burkholderia pseudomallei/genética , Ceftazidima/farmacologia , Melioidose/diagnóstico , Hidrólise , Mutação , Ribonucleases
8.
BMC Infect Dis ; 23(1): 672, 2023 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-37814214

RESUMO

BACKGROUND: Remdesivir is considered to be a specific drug for treating coronavirus disease 2019. This systematic review aims to evaluate the clinical efficacy and risk of remdesivir alone and in combination with other drugs. RESEARCH DESIGN AND METHODS: The PubMed, Embase, SCIE, Cochrane Library, and American Clinical trial Center databases were searched up to 1 April 2022 to identify. Randomized controlled trials (RCTs) and observational studies comparing the efficacy of remdesivir monotherapy and combination therapy with that of control drugs. RESULTS: Ten RCTs and 32 observational studies were included in the analysis. Regarding the primary outcome, remdesivir use reduced mortality in patients with severe COVID-19 (RR = 0.57, 95% CI (0.48,0.68)) and shortened the time to clinical improvement (MD = -2.51, 95% CI (-2.75, -2.28)). Regarding other clinical outcomes, remdesivir use was associated with improved clinical status (RR = 1.08, 95%CI (1.01, 1.17)). Regarding safety outcomes, remdesivir use did not cause liver or kidney damage (RR = 0.87, 95%CI (0.68, 1.11)) (RR = 0.88, 95%CI (0.70,1.10)). Compared with remdesivir alone, remdesivir combined with other drugs (e.g., steroids, favipiravir, and convalescent plasma) had no effect on mortality. CONCLUSION: The use of remdesivir can help to reduce the mortality of patients with severe COVID-19 and shorten the time to clinical improvement. There was no benefit of remdesivir combination therapy for other clinical outcomes. TRIAL REGISTRATION: PROSPERO registration number: CRD42022322859.


Assuntos
COVID-19 , Humanos , SARS-CoV-2 , Soroterapia para COVID-19 , Tratamento Farmacológico da COVID-19 , Resultado do Tratamento
9.
China CDC Wkly ; 5(37): 822-828, 2023 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-37814632

RESUMO

China's six tropical regions include Guangdong Province, Yunnan Province, Hainan Province, Hong Kong Special Administrative Region (SAR), Macau SAR, and Taiwan, China. Hainan, seated in the southernmost tropical region of China, is home to ticks that remain active throughout all four seasons. This heightens their potential to transmit tick-borne diseases to both animals and humans. This study provides a succinct overview of the prevailing tick species' spatial distribution and offers an outline of the range and dispersion of emerging tick-borne infections in tick vectors, animal hosts, and human populations within Hainan, China.

10.
Biochem Biophys Res Commun ; 682: 397-406, 2023 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-37852065

RESUMO

TssJ-3 is an outer-membrane lipoprotein and is one of the key components of the type VI secretion system in Burkholderia pseudomallei. TssJ translocates effector proteins to target cells to induce innate immune response in the host. However, the tssJ gene has not been identified in B. pseudomallei and its function in this bacterium has not yet been characterized. tssJ-3 knockout and tssJ-3-complemented B. pseudomallei strains were constructed to determine the effects of tssJ-3 on bacterial growth, biofilm formation, flagellum synthesis, motility, host cell infection, and gene expression in B. pseudomallei. We found that the ΔtssJ-3 mutant strain of B. pseudomallei showed significantly suppressed biofilm formation, flagellum synthesis, bacterial growth, motility, and bacterial invasion into host cells (A549 cells). Furthermore, the ΔtssJ-3 mutation downregulated multiple key genes, including biofilm and flagellum-related genes in B. pseudomallei and induced interleukin-8 gene expression in host cells. These results suggest that tssJ-3, an important gene controlling TssJ-3 protein expression, has regulatory effects on biofilm formation and flagellum synthesis in B. pseudomallei. In addition, B. pseudomallei-derived tssJ-3 contributes to cell infiltration and intracellular replication. This study provides a molecular basis of tssJ-3 for developing therapeutic strategies against B. pseudomallei infections.


Assuntos
Burkholderia pseudomallei , Melioidose , Sistemas de Secreção Tipo VI , Humanos , Burkholderia pseudomallei/genética , Virulência/genética , Melioidose/microbiologia , Sistemas de Secreção Tipo VI/genética , Sistemas de Secreção Tipo VI/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo
11.
Chemosphere ; 340: 139814, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37586486

RESUMO

The toxicity of Polystyrene (PS) may be higher through co-exposure with other pollutants. Human can simultaneously face the challenges from the various pollutants. Nevertheless, little research has been done on the combined effects of PS and 2,6-dichloro-p-benzoquinone (DCBQ) disinfection byproduct. Considering the potential risk of PS and DCBQ, we aimed to illustrate the effects of PS in combination with DCBQ on the immune responses of mice. We found that cotreatment of DCBQ and PS may inhibit the activity of spleen CD4+ T cells and interfere with the normal function of the immune system. Further research found that DCBQ + PS resulted in increasing amount of the inflammatory cells in intestine via histopathological evaluation. The reason might be that DCBQ + PS has changed the composition of intestinal flora, abnormally activated intestinal macrophage, and inhibited the expression of immune-related genes, thus leading to intestinal immune disorders and triggering intestinal inflammation. In summary, PS may alter the cooperation mechanism of gut microbiota and immune system through co-exposure with DCBQ. Current results suggested that more attention should be paid to the combined toxic effects of environmental contaminants.


Assuntos
Poluentes Ambientais , Microbioma Gastrointestinal , Poluentes Químicos da Água , Humanos , Animais , Camundongos , Poliestirenos/toxicidade , Benzoquinonas/toxicidade , Sistema Imunitário , Poluentes Químicos da Água/análise
12.
Front Microbiol ; 14: 1190939, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37455722

RESUMO

Since the pandemic started, the coronavirus disease 2019 (COVID-19) has spread worldwide. In patients with COVID-19, the gut microbiome (GM) has been supposed to be closely related to the progress of the disease. The gut microbiota composition and human genetic variation are also connected in COVID-19 patients, assuming a triangular relationship between the genome, GM, and COVID-19. Here, we reviewed the recent developments in the study of the relationship between gut microbiota and COVID-19. The keywords "COVID-19," "microbiome," and "genome" were used to search the literature in the PubMed database. We first found that the composition of the GM in COVID-19 patients varies according to the severity of the illness. Most obviously, Candida albicans abnormally increased while the probiotic Bifidobacterium decreased in severe cases of COVID-19. Interestingly, clinical studies have consistently emphasized that the family Lachnospiraceae plays a critical role in patients with COVID-19. Additionally, we have demonstrated the impact of microbiome-related genes on COVID-19. Specially, we focused on angiotensin-converting enzyme 2's dual functions in SARS-CoV-2 infection and gut microbiota alternation. In summary, these studies showed that the diversity of GMs is closely connected to COVID-19. A triangular relationship exists between COVID-19, the human genome, and the gut flora, suggesting that human genetic variations may offer a chance for a precise diagnosis of COVID-19, and the important relationships between genetic makeup and microbiome regulation may affect the therapy of COVID-19.

13.
Folia Parasitol (Praha) ; 702023 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-37326358

RESUMO

Ticks are important vectors of various pathogens that cause infectious diseases in humans. Endosymbiotic bacteria have been explored as targets for tick and tick-borne disease control. However, the tick bacterial community on Hainan Island, which is the largest tropical island in China and has an environment favourable to ticks, has not yet been studied. In this study, we surveyed the bacterial community of ticks collected from grass in one village in Haikou. A total of 20 ticks were morphologically and molecularly identified as Haemaphysalis spp. The tick bacterial 16S rRNA hypervariable region amplicon libraries were sequenced on an Illumina MiSeq platform. A total of 10 possible bacterial genera were detected, indicating a low-diversity bacterial community profile. The dominant bacterial genus, Massilia, accounted for 97.85% of the population. Some other bacterial genera, including Arsenophonus and Pseudomonas, have been reported to play a role in tick development and tick-borne pathogen transmission in other tick species. Overall, the study highlights the first descriptive understanding of the tick bacterial community on Hainan Island and provides a basis for deciphering the interactions between the tick microbiome and tick-borne pathogens.


Assuntos
Ixodidae , Carrapatos , Animais , Humanos , RNA Ribossômico 16S/genética , Ixodidae/microbiologia , Bactérias/genética , China/epidemiologia
14.
Cytogenet Genome Res ; 163(1-2): 59-73, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37385223

RESUMO

Ticks are hematophagous arthropods and obligate ectoparasites of humans and other animals. This study focused on the molecular discrimination of ticks in the tropical environment of Hainan according to multi-gene DNA barcode markers with the expectation of accurately distinguishing species. A total of 420 ticks, including 49 adult ticks, 203 nymphal ticks, and 168 larval ticks, were collected in the field, and the 49 adult ticks were identified as Rhipicephalus turanicus, Dermacentor marginatus, and Haemaphysalis longicornis. The mitochondrial 16S rRNA, ribosomal 28S rRNA D2, and ribosomal internal transcribed spacer 2 (ITS2) regions were used as DNA barcode markers to discriminate species. According to basic local alignment search tool analysis against the GenBank database, 16S rRNA positively identified ticks in the Rhipicephalus, Dermacentor, and Haemaphysalis genera; the 28S rRNA D2 region identified ticks in the Rhipicephalus and Dermacentor genera; and ITS2 identified ticks as D. marginatus. Pairwise sequence comparisons based on these three regions were visualized with a Sequence Demarcation Tool matrix. Substitution saturation tests using data analysis and molecular biology and evolution revealed little substitution saturation (Iss < Iss.c, p < 0.05) in the 16S rRNA region for the Haemaphysalis genus; 28S rRNA D2 region for the Rhipicephalus, Dermacentor, and Haemaphysalis genera; and ITS2 region for the Rhipicephalus and Dermacentor genera. Distinctive sequences for which it is difficult to obtain good matches with the sequences available in GenBank exist in the ticks of Hainan. Future studies should obtain complementary sequences to refine and update the database for the molecular characterization of ticks.


Assuntos
Ixodidae , Rhipicephalus , Animais , Humanos , Ixodidae/genética , RNA Ribossômico 16S/genética , RNA Ribossômico 28S , Código de Barras de DNA Taxonômico , Rhipicephalus/genética , Marcadores Genéticos , China , Variação Genética/genética , Filogenia
15.
Virus Res ; 332: 199138, 2023 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-37211158

RESUMO

A newly discovered tick-borne virus called the severe fever with thrombocytopenia syndrome virus (SFTSV) can cause the severe fever with thrombocytopenia syndrome (SFTS). The mortality and incidence rate of SFTS patients remain extremely high due to the fast global dissemination of its arthropod vectors, and the mechanism of viral pathogenesis remains largely unknown. In this study, high-throughput RNA sequencing (RNA-Seq) was used to sequence HEK 293 cells treated with SFTSV at four time points. 115, 191, 259, and 660 differentially expressed genes (DEGs) were identified at 6, 12, 24, and 48 h post-infection, respectively. We found that SFTSV infection induced the expression of genes responsible for numerous cytokine-related pathways, including TNF, CXCL1, CXCL2, CXCL3, CXCL8, CXCL10, and CCL20. With the extension of infection time, the expression of most genes involved in these pathways increased significantly, indicating the host's inflammatory response to SFTSV. Moreover, the expression levels of GNA13, ARHGEF12, RHOA, ROCK1, and MYL12A, elements of the platelet activation signaling pathway, were downregulated during SFTSV infection, suggesting that the SFTSV infection may cause thrombocytopenia by inhibiting platelet activation. Our results contribute to further understanding the interaction between SFTSV and the host.


Assuntos
Infecções por Bunyaviridae , Phlebovirus , Febre Grave com Síndrome de Trombocitopenia , Humanos , Células HEK293 , Phlebovirus/genética , Transdução de Sinais , Quinases Associadas a rho/metabolismo
16.
Microbiome ; 11(1): 101, 2023 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-37158937

RESUMO

BACKGROUND: The pathogenic viruses transmitted by mosquitoes cause a variety of animal and human diseases and public health concerns. Virome surveillance is important for the discovery, and control of mosquito-borne pathogenic viruses, as well as early warning systems. Virome composition in mosquitoes is affected by mosquito species, food source, and geographic region. However, the complex associations of virome composition remain largely unknown. RESULTS: Here, we profiled the high-depth RNA viromes of 15 species of field-caught adult mosquitoes, especially from Culex, Aedes, Anopheles, and Armigeres in Hainan Island from 2018 to 2020. We detected 57 known and 39 novel viruses belonging to 15 families. We established the associations of the RNA viruses with mosquito species and their foods, indicating the importance of feeding acquisition of RNA viruses in determining virome composition. A large fraction of RNA viruses were persistent in the same mosquito species across the 3 years and different locations, showing the species-specific stability of viromes in Hainan Island. In contrast, the virome compositions of single mosquito species in different geographic regions worldwide are visibly distinct. This is consistent with the differences in food sources of mosquitoes distributed broadly across continents. CONCLUSIONS: Thus, species-specific viromes in a relatively small area are limited by viral interspecific competition and food sources, whereas the viromes of mosquito species in large geographic regions may be governed by ecological interactions between mosquitoes and local environmental factors. Video Abstract.


Assuntos
Aedes , Anopheles , Culex , Humanos , Adulto , Animais , Viroma/genética , Alimentos
17.
Integr Zool ; 18(6): 1009-1013, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36905201

RESUMO

Severe fever with thrombocytopenia syndrome virus (SFTSV) RNA level increased in female ticks after injection with SFTSV. Furthermore, SFTSV RNA was detected in the eggs and larvae that originated from the virus-infected female ticks.


Assuntos
Infecções por Bunyaviridae , Phlebovirus , Rhipicephalus sanguineus , Febre Grave com Síndrome de Trombocitopenia , Animais , Feminino , Rhipicephalus sanguineus/genética , Phlebovirus/genética , China , RNA
18.
Biosens Bioelectron ; 225: 115098, 2023 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-36701949

RESUMO

In this work, PtPd nanoparticles functionalized porphyrin metal-organic framework nanoenzymes (PtPd@PCN-224 nanoenzymes) are exploited as signal amplification tags to fabricate a rapid and ultrasensitive sensitive CRISPR/Cas14a-based electrochemical biosensor for Burkholderia pseudomallei (B. pseudomallei) specific DNA sequences detection. The prepared PtPd@PCN-224 nanoenzymes not only catalyze the reduction peak current of H2O2 to obtain a strong electrochemical signal output, but also provide massive active sites for the assembly of nucleic acids by Zr-O-P bonds. Besides, the designed target-activated CRISPR/Cas14a is able to recognize the target DNA sequences and further trigger the trans-cleavage of ssDNA for signal amplification. Benefiting from the target-activated CRISPR/Cas14a and PtPd@PCN-224 nanoenzymes, the developed electrochemical biosensor for B. pseudomallei DNA detection exhibits high sensitivity with detection of limit down to 12.8 aM and excellent specificity for distinguishing non-targeted bacteria. Moreover, the CRISPR/Cas14a-based electrochemical detection platform can also apply for other pathogenic bacteria diagnostic by well-designing sgRNA for target sequence recognition, possessing high flexibility and versatility in clinical diagnosis.


Assuntos
Técnicas Biossensoriais , Burkholderia pseudomallei , Burkholderia pseudomallei/genética , Peróxido de Hidrogênio/química , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Técnicas Eletroquímicas
19.
Biosens Bioelectron ; 222: 114976, 2023 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-36516632

RESUMO

Electrocheluminescence analysis amplified by coreaction accelerators has experienced breakthrough in ultrasensitive detection of biomarkers. Herein, a highly efficient coreaction accelerator, two-dimensional layered MoSe2 nanosheets loaded with gold nanoparticles (AuNPs@MoSe2 heterostructure), is proposed to enhance the ECL efficiency of Ru(bpy)32+/tripropylamine (TPrA) system. The presence of AuNPs avoids the aggregation of MoSe2 nanosheets, and improves the electrical conductivity of modified surface. The AuNPs@MoSe2 modified electrode also provides a large area for loading of abundant capture probe. MoSe2 as an electroactive substrate can remarkably accelerate the generation of TPrA•+ radicals to react with electrooxidized Ru(bpy)32+, which achieves about 3.4-fold stronger ECL intensity. Thus, an enhanced ECL immunoassay method can be achieved after Ru(bpy)32+-doped silica nanoparticle labeled antibody (Ab2-Ru@SiO2) is captured to the modified electrode via immunological recognition. Using methylated DNA as a target, the immunosensor was prepared by binding capture DNA on AuNPs@MoSe2 modified electrode to successively capture the target, anti-5-methylcytosine antibody (anti-5mC) and Ab2-Ru@SiO2. The proposed strategy could detect 0.26 fM 5 mC (3σ) with a detectable concentration range of 1.0 fM - 10 nM at methylated DNA. This immunosensor showed excellent selectivity, good stability and reproducibility, and acceptable recovery, indicating the broad prospects of the novel coreaction accelerator in clinical diagnosis.


Assuntos
Técnicas Biossensoriais , Nanopartículas Metálicas , Nanopartículas Metálicas/química , Ouro/química , Dióxido de Silício/química , Técnicas Biossensoriais/métodos , Metilação de DNA , Reprodutibilidade dos Testes , Imunoensaio/métodos , Técnicas Eletroquímicas/métodos , Medições Luminescentes
20.
China Tropical Medicine ; (12): 347-2023.
Artigo em Chinês | WPRIM (Pacífico Ocidental) | ID: wpr-979684

RESUMO

@#Abstract: Objective To investigate the composition and diversity of midgut microbial community of Haemaphysalis longicornis infected with severe fever with thrombocytopenia syndrome virus (SFTSV). Methods The midgut DNA of three group Haemaphysalis longicornis infected with SFTSV was extracted, and the 16S rDNA gene of the sample was sequenced by HiSeq platform. The composition and diversity of endosymbiotic microbial community were clarified by OTU cluster analysis and alpha diversity analysis. Results The midgut microbial clusters of the three groups infected with SFTSV were 143, 113, 163 OTUs respectively; the sparsity curve and abundance grade curve showed that the data had sufficient sequencing depth, and the midgut of Haemaphysalis longicornis infected with SFTSV was rich in microbial composition, but the species distribution was uneven. The analysis of microbial community composition showed that Proteobacteria, Firmicutes and Actinobacteria were the main dominant bacteria at the phyla level. At the class level, Gammaproteobacteria, Bacilli, Betaproteobacteria and Actinomycetia were the main dominant bacteria. At the order level, Legionellales, Bacillales, Burkholderiales and Actinomycetales were the main dominant orders. At the family level, Coxiellaceae, Bacillaceae, Moraxellaceae and Rhodococcaceae were the main dominant families. At the genus level, the relative abundance of Coxiella was the highest, followed by Aeribaillus and Azonexus. Alpha diversity analysis showed that the average Shannon index was 139.67, the average Simpson index was 0.48, the average Chao index was 145.06, and the average ACE index was 147.11. Conclusions The species diversity of intestinal microorganisms in Haemaphysalis longicornis infected with SFTSV is rich. The results provide a basis for further exploring the interaction between intestinal microbes of Haemaphysalis longicornis and SFTSV and developing new ideas for the prevention and control of ticks and tick-borne diseases.

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