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Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(8): 695-701, 2019 Aug.
Artigo em Chinês | MEDLINE | ID: mdl-31638566


Objective To accurately and rapidly detect and type five classical Staphylococcal enterotoxins (SEs) by array-ELISA using a combination of a chip and ELISA. Methods SEs were prepared by prokaryotic expression and affinity chromatography. Hybridoma cells were injected intraperitoneally into mice to prepare ascites. A monoclonal antibody was obtained by ascites purification. The sensitivity and specificity of the antibody were evaluated by ELISA. The antibody was printed in one cell, and the sensitivity and specificity of array-ELISA were evaluated. Results Except for the detection limit of Staphylococcal enterotoxin C (SEC) being 10 ng/mL, 0.0001 ng/mL SEs could be detected by array-ELISA in PBS. The detection limit was 0.001-10 ng/mL for SEs in milk. The specificity was 100% in both PBS and milk. No cross reaction was observed between SEs. Additionally, no cross reaction was observed between SEB and botulinum toxin. Conclusion Array-ELISA has been successfully established, and it can simultaneously detect and discriminate five classical SEs within one sample sensitively and specifically.

Toxicon ; 165: 62-68, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31047932


Staphylococcal enterotoxin B (SEB) is an important enterotoxin which is a major reason for food poisoning and a potential biologic agent. Hence, rapid and accurate detection is very important. An amplified luminescent proximity homogeneous assay (AlphaLISA) for SEB detection was established here. Its performance was evaluated on mock specimen and culture supernatant. Its free from the effect of protein A was compared with ELISA. Results showed that the linear range for SEB detection was 25 pg/mL to 25 ng/mL. The detection limitation is 25 pg/mL in buffer and 50 pg/mL in specimen respectively. There was no cross-reaction with other classical SEs or botulinum toxin. AlphaLISA was also tolerant to the matrix of sample and showed good repeatability. The inter-assay coefficient of variation (CV) and intra-assay CV were<10% for buffer and mock specimens, respectively. Besides AlphaLISA detection was free of the interference of protein A (which is the obstacle for immune-based detection of SEs in Staphylococcus aureus culture supernatants): this feature is very important for food-poisoning confirmation caused by SEB contamination. These data suggest that the AlphaLISA established here is well suited for SEB detection in food samples and S. aureus culture supernatants.

Enterotoxinas/análise , Imunoensaio/métodos , Enterotoxinas/química , Contaminação de Alimentos/análise , Limite de Detecção , Proteína Estafilocócica A/química
Sci Rep ; 8(1): 12828, 2018 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-30150783


Bloodstream infections (BSIs) are often life-threatening, and rapid identification is critical. Here, we developed a TaqMan array card (TAC) assay to detect pathogens in BSI specimens. The TAC included 30 primer/probe pairs targeting 27 species and 3 controls. Reverse transcription and 0.1% blue dextran 2000 increased the TAC assay efficiency. The primer/probe pairs had a limit of detection of 100-102 CFU/mL and a specificity of 100%. For whole blood specimens, the TAC assay showed a sensitivity and specificity of 79.4% and 99.69%, respectively. For blood culture samples, the TAC assay showed a sensitivity and specificity of 100% and 99.67%, respectively. The TAC assay could be a promising method for early detection of bloodstream infection.

Virulence ; 8(7): 1290-1302, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-28402705


Streptococcus suis is an important emerging zoonotic agent that causes acute bacterial meningitis in humans with high mortality and morbidity. Our previous work showed that factor H-binding protein (Fhb) contributed to virulence of S. suis, but the role of Fhb in the development of S. suis meningitis remained unclear. In this study, we demonstrated for the first time that Fhb contributed to the traversal of S. suis across the human blood-brain barrier by allelic-exchange mutagenesis, complementation and specific antibody blocking studies. We also showed that globotriaosylceramide (Gb3), the receptor of Fhb, was involved in this process and affected S. suis infection-induced activation of myosin light chain 2 through Rho/ROCK signaling in hCMEC/D3 cells. Using a murine model of S. suis meningitis, we further demonstrated that Gb3-deficiency prevented the mice from developing severe brain inflammation or injury. Our results demonstrate that the Fhb-Gb3 interaction plays an important role in the development of S. suis meningitis and might be a potential therapeutic target against S. suis infection.

Proteínas de Bactérias/metabolismo , Meningites Bacterianas/metabolismo , Infecções Estreptocócicas/metabolismo , Streptococcus suis/metabolismo , Triexosilceramidas/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Transporte , Feminino , Interações Hospedeiro-Patógeno , Humanos , Masculino , Meningites Bacterianas/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Infecções Estreptocócicas/microbiologia , Streptococcus suis/genética , Streptococcus suis/patogenicidade , Virulência
FEBS Lett ; 590(9): 1384-92, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-27086582


The recently identified Streptococcus suis adhesin factor H-binding protein (Fhb) targets the host cellular receptor glycolipid GbO3 through its N terminus. However, it is unclear how Fhb interacts with its receptor. Here, we determined the complex structure of factor H-binding protein receptor-binding domain (Fhb RBD) with Gb2, an analog of its receptor, revealing that Gb2 binds in a pocket of the ß sandwich core domain. We identified the key residues for Fhb RBD receptor binding using mutagenesis and isothermal titration calorimetry. Mutagenesis analyses indicated that Fhb binds to Gb2 mainly through hydrogen and hydrophobic interactions. Our findings provided structural insights into the Fhb-mediated host-pathogen interactions of S. suis.

Adesinas Bacterianas/química , Streptococcus suis/química , Triexosilceramidas/metabolismo , Adesinas Bacterianas/genética , Adesinas Bacterianas/metabolismo , Sítios de Ligação , Humanos , Mutação Puntual , Ligação Proteica , Streptococcus suis/patogenicidade , Triexosilceramidas/química
Front Microbiol ; 6: 1001, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26441928


Muramidase-released protein (MRP) is as an important virulence marker of Streptococcus suis (S. suis) serotype 2. Our previous works have shown that MRP can bind human fibrinogen (hFg); however, the function of this interaction in S. suis meningitis is not known. In this study, we found that the deletion of mrp significantly impairs the hFg-mediated adherence and traversal ability of S. suis across human cerebral microvascular endothelial cells (hCMEC/D3). Measurement of the permeability to Lucifer yellow in vitro and Evans blue extravasation in vivo show that the MRP-hFg interaction significantly increases the permeability of the blood-brain barrier (BBB). In the mouse meningitis model, wild type S. suis caused higher bacterial loads in the brain and more severe histopathological signs of meningitis than the mrp mutant at day 3 post-infection. Western blot analysis and immunofluorescence observations reveal that the MRP-hFg interaction can destroy the cell adherens junction protein p120-catenin of hCMEC/D3. These results indicate that the MRP-hFg interaction is important in the development of S. suis meningitis.